JP2022539584A - Methods and compositions for treatment of pancreatic cancer - Google Patents

Abstract

The present invention provides various compositions and methods useful in the treatment of pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), and methods for activating pancreatic tissue-specific anti-tumor T-cell immunity. In some embodiments, such methods comprise administration of IL33. In some embodiments, such methods comprise administration of PD-1 and/or PD-L1 inhibitors.

Description

RELATED APPLICATION CROSS REFERENCE This application is based on U.S. Provisional Patent Application No. 62/868,976 filed June 30, 2019 and U.S. Provisional Patent Application No. 62/937,219 filed November 18, 2019 No. 1, 2003, the content of each of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on June 30, 2020, is called MSKCC_042_WO1_SL.txt and is 15,983 bytes in size.

INCORPORATION BY REFERENCE All documents cited herein are hereby incorporated by reference in their entirety under any statute that permits inclusion by reference. Also incorporated by reference are the manufacturer's instructions or catalogs for products cited or referred to herein. Documents incorporated herein by reference, or any teachings therein, may be used in the practice of the present invention. Numbers in superscripts following text in this specification refer to the numbered publications identified in the "List of References" section of this patent application.

BACKGROUND Antibodies that target the PD-1/PD-L1 and CTLA-4 pathways are useful as cancer immunotherapies. However, most cancers lack pre-existing anti-tumor T cells and are unresponsive. Pancreatic ductal adenocarcinoma (PDAC) is one of the most immunotherapy-resistant and deadly cancers. Therefore, there is an urgent need for new and improved methods of treating PDAC, including immunotherapy-resistant PDAC. The present invention fulfills this need.

SUMMARY OF THE INVENTION The present invention is based, in part, on a series of important discoveries that are described in more detail in the Examples herein. Briefly, using a unique cohort of long-term survivors of pancreatic ductal adenocarcinoma (PDAC), tumor expression of group 2 innate lymphocytes (ILC2) and the ILC2-activating ligand interleukin (IL)-33 was significantly increased in tumors. It was found to positively correlate with immune cytolytic activity and long-term patient survival. Using the PDAC mouse model, the IL33-ILC2 axis was found to activate pancreatic tissue-specific anti-tumor T-cell immunity. Surprisingly, it was found that recombinant IL33 (rIL33) activates PDAC TILC2 and CD8 ⁺ T cells and cures over 70% of mice tested. Furthermore, combined rIL33 treatment and PD-1/PD-L1 pathway blockade (using αPD-1) synergized pancreatic tissue-specific ILC2 in both PD-1 partially sensitive and PD-1 resistant models. It was found that the antitumor effect of αPD-1 was enhanced. Importantly, in a highly aggressive PD-1-resistant tumor model, treatment with rIL33 or αPD-1 alone had limited efficacy, whereas the combination of rIL33 and αPD-1 reduced all other treatment resulted in significantly smaller tumors, with approximately a 40% reduction in tumor volume and a 50% improvement in survival. Based on these discoveries, and others described herein, the present invention provides various new and improved compositions and methods for the treatment of pancreatic cancer.

For example, in one aspect, the invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering to the subject having PDAC an effective amount of IL33, thereby treating PDAC in said subject.

In another aspect, the present invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising an effective amount in a subject having PDAC of: (a) IL33 and (b) PD-1 and/or administering a PD-L1 inhibitor, thereby treating PDAC in a subject.

In yet another aspect, the invention provides a method of activating pancreatic tissue-specific anti-tumor T-cell immunity in a subject in need thereof, comprising administering an effective amount of IL33 to a subject with PDAC, thereby A method is provided comprising activating tissue-specific anti-tumor T cell immunity in a subject. In some such aspects, activation of pancreatic tissue-specific anti-tumor T cell immunity comprises activation/proliferation of pancreatic ILC2 cells and/or activation of CD8 ⁺ T cells.

In another aspect, the invention provides a method of activating pancreatic tissue-specific anti-tumor T-cell immunity in a subject in need thereof, comprising administering to a subject having PDAC an effective amount of (a) IL33 and (b) and a PD-1 and/or PD-L1 inhibitor, thereby activating pancreatic tissue-specific anti-tumor T cell immunity in the subject. In some such aspects, activation of pancreatic tissue-specific anti-tumor T cell immunity comprises activation/proliferation of pancreatic ILC2 cells and/or activation of CD8 ⁺ T cells.

In another aspect, the invention provides a method of activating pancreatic ILC2 cells, comprising contacting the pancreatic ILC2 cells with an effective amount of IL33, thereby activating the pancreatic ILC2 cells. .

In another aspect, the invention provides a method of activating pancreatic ILC2 cells, comprising contacting the pancreatic ILC2 cells with an effective amount of (a) IL33 and (b) a PD-1 and/or PD-L1 inhibitor, A method is provided comprising thereby activating pancreatic ILC2 cells.

In another aspect, the invention provides a method of sensitizing PDAC tumor and/or pancreatic ILC2 cells to PD-1 and/or PD-L1 inhibitors, wherein the PDAC tumor and/or pancreatic ILC2 cells are effectively A method is provided comprising contacting with an amount of IL33, thereby sensitizing PDAC tumor and/or pancreatic ILC2 cells to PD-1 and/or PD-L1 inhibitors.

In some aspects, the invention provides compositions comprising (a) IL33 and (b) a PD-1 and/or PD-L1 inhibitor. For example, in some embodiments, the invention provides compositions for use in treating PDAC comprising (a) IL33 and (b) a PD-1 and/or PD-L1 inhibitor.

In some embodiments, the methods summarized above or described elsewhere herein determine whether a subject's tumor and/or pancreas contains IL-33 receptor-expressing ILC2 cells. or determining whether the ILC2 cells express the IL-33 receptor. In some such embodiments, the IL33 and/or PD-1/PD-L1 inhibitor is administered to the subject only if the subject's tumor and/or pancreas comprises ILC2 cells that express the IL-33 receptor. administered. Similarly, in some such embodiments, the ILC2 cells are contacted with IL33 and/or the PD-1/PD-L1 inhibitor only if the ILC2 cells express the IL-33 receptor.

In some aspects, the present invention provides various cell therapy methods. For example, in one aspect, the invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering to a recipient subject having PDAC an effective amount of activated donor pancreatic ILC2 cells. wherein the donor pancreatic ILC2 cells are obtained from a donor subject and have been activated ex vivo/in vitro by contact with IL33, and the donor and recipient subjects are allogeneic, whereby the recipient subject provided is a method comprising treating PDAC in

Similarly, in another aspect, the invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising: (a) treating donor pancreatic ILC2 cells obtained from a donor subject ex vivo with IL33; / contacting in vitro to generate activated donor pancreatic ILC2 cells; and (b) administering the donor activated pancreatic ILC2 cells to a recipient subject with pancreatic ductal adenocarcinoma (PDAC), wherein the donor subject and recipe The recipient subject is allogeneic, thereby providing a method comprising treating PDAC in a recipient subject.

Similarly, in yet another aspect, the invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof comprising: (a) obtaining donor pancreatic ILC2 cells from a donor subject; a) ex vivo/in vitro contacting of donor pancreatic ILC2 cells with IL33 to generate activated donor pancreatic ILC2 cells; wherein the donor subject and the recipient subject are allogeneic, thereby treating PDAC in the recipient subject.

In some embodiments, the cell therapy methods described above or elsewhere herein also include the use of donor pancreatic ILC2 cells and/or activated donor pancreatic ILC2 cells prior to administering the activated donor pancreatic ILC2 cells to a recipient subject. Also included is the step of expanding the donor pancreatic ILC2 cells ex vivo/in vitro. In some of the cell therapy methods described above or elsewhere herein, the donor pancreatic ILC2 cells administered to the recipient subject are a substantially pure population of ILC2 cells. Such a substantially pure population of ILC2 cells may be isolated by any suitable cell isolation/purification method, such as one or more pancreatic ILC2 markers (such as those described in the Examples section of this patent application). ) can be obtained by cell sorting based on the presence of In some of the cell therapy methods described above or elsewhere herein, the donor subject and recipient subject are the same individual, such that the method is an autologous cell therapy method. Similarly, in some of the cell therapy methods described above or elsewhere herein, the donor and recipient subjects have the same MHC/HLA types.

In some of the embodiments summarized above or described elsewhere herein, the subject (including, in the case of cell therapy methods, a donor subject and/or a recipient subject) is a human. In some of the embodiments summarized above or described elsewhere herein, the subject (including, in the case of cell therapy methods, donor and/or recipient subjects) is a non-human mammal. In some of the embodiments summarized above or described elsewhere herein, the subject (including donor embodiments and/or recipient subjects in the case of cell therapy methods) is a mouse. In some of the embodiments summarized above or described elsewhere herein, the subject (including, in the case of cell therapy methods, a donor subject and/or a recipient subject) is PD-1 and/or PD - Have PDAC that is partially or completely resistant to L1 inhibitor treatment.

In some of the aspects summarized above or described elsewhere herein, the IL33 is recombinant IL33. In some of the aspects summarized above or described elsewhere herein, IL33 is human IL33. In some of the aspects summarized above or described elsewhere herein, the IL33 is recombinant human IL33. In some of the aspects summarized above or described elsewhere herein, the IL33 is murine IL33. In some of the aspects summarized above or described elsewhere herein, the IL33 is recombinant murine IL33.

Among those embodiments summarized above or described elsewhere herein that include a PD-1 inhibitor, in some such embodiments, the PD-1 inhibitor is an antibody. In some of the embodiments summarized above or described elsewhere herein, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, semiplimab, AMP-224, AMP-514 and PDR001. be.

In some of the embodiments summarized above or described elsewhere herein that comprise a PD-L1 inhibitor, in some of such embodiments the PD-L1 inhibitor is an antibody is. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559 and CK-301.

These and other aspects of the present invention are further described in the Detailed Description, Figures and Examples section of this patent application. Moreover, those skilled in the art will recognize that the various aspects of the invention described throughout this patent application can be combined in various different ways and such combinations are within the scope of the invention. can.

Figure la-f: IL33-dependent ILC2 infiltrates human and mouse pancreatic cancer. (Fig. 1a) Gating, frequency and phenotype of ILCs in unselected human PDAC patients. (Fig. 1b) Frequency of ILC2 in tumor tissue microarrays of short- and long-term PDAC survivors (top) and association with survival time (bottom). (Fig. 1c) Short- and long-term PDAC survivor bulk tumor IL33 mRNA association with survival and correlation with tumor lytic index (CYT). (Fig. 1d) Gating and frequency of ILCs in PDAC mice. (FIG. 1e) Frequency and number of intratumoral ILCs in Rag2 ^−/− PDAC mice treated with αCD90.2 or isotype (Iso) antibody. (Fig. 1f) Gating, frequency and number of ILCs in Il33 ^+/+ and Il33 ^−/− PDAC mice. High and Low in FIG. 1b and Low in FIG. 1c defined above and below the cohort median, respectively. Data were collected 14 days after tumor implantation (FIG. 1d-f). n, number of tumors from individual patients or mice. Horizontal bars indicate median values. Data in FIGS. 1d-f were pooled from ≧2 independent experiments with n≧3/group, each point representing one mouse analyzed separately. P-values are expressed by Tukey's one-way ANOVA (Fig. 1a) and Kruskal-Wallis post-multiple comparison test (Fig. 1d), two-tailed Mann-Whitney test (Fig. 1b, e, f), two-tailed log-rank (Fig. 1b, c, survival curve), and determined by linear regression (Fig. 1c). Figure 2a-g: The IL33-ILC2 axis activates tissue-specific cancer immunity. Tumor weight, volume and survival of Il33 ^+/+ and Il33 ^−/− orthotopic (Fig. 2a) or subcutaneous (Fig. 2b) PDAC mice. (Fig. 2c) Frequencies of total CD8 ⁺ T cells (left) and IFN-producing (right) cells in orthotopic Il33 ^+/+ and Il33 ^−/− PDAC tumors. (Fig. 2d) Tumor weights of T-cell depleted Il33 ^+/+ and Il33 ^−/− orthotopic PDAC mice. (Fig. 2e) Frequency of tumor rejection and tumor weight in Il33 ^+/+ and Il33 ^−/− orthotopic and subcutaneous KPC-OVA PDAC mice. (Fig. 2f) Study design of KPC-OVA PDAC tumors in iCOS-T mice with intact or depleted ILC2 (left), frequency of tumor rejection (middle) and tumor weight (right). (Fig. 2g) Frequency of OVA-specific CD8 ⁺ T cells in the draining lymph nodes of orthotopic KPC-OVA PDAC iCOS-T mice with intact or depleted ILC2. Data were collected 14 days (Fig. 2a,c,d), 28 days (Fig. 2b), 42 days (Fig. 2e) and 8 days (Fig. 2f,g) after transplantation. Horizontal bars indicate medians, error bars indicate s.e.m. Data are pooled from two or more independent studies with n≧4/group, n and data points indicate individual mice analyzed separately. P values are two-tailed Mann-Whitney test (Fig. 2a-g), two-tailed log-rank test (Fig. 2a,b, survival curves), two-way ANOVA with Sidak's multiple comparison test (Fig. 2a,b, tumor volume ) and chi-square test (Fig. 2e, f% rejection). Figure 3a-h: ILC2 stimulates tissue-specific cancer immunity by recruiting intratumoral dendritic cells. (Fig. 3a) Tumor weight, volume and survival of orthotopic and subcutaneous PDAC mice treated with vehicle or recombinant IL33 (rIL33). (Fig. 3b) Tumor weight and volume of orthotopic and subcutaneous PDAC mice treated with vehicle or recombinant IL18 (rIL18). (Fig. 3c) Gating, frequency and number of ILC2 in orthotopic PDAC mice treated with rIL33 (DLN vehicle, n=13; tumor vehicle, n=12). (Fig. 3d) Gating and frequency of CD103 ⁺ dendritic cells (DC) in tumors of orthotopic PDAC mice treated with rIL33. (Fig. 3e) Tumor weight, volume and (Fig. 3f) frequency of CD103 ⁺ DC in tumors of wild-type (WT) and ILC2-deficient orthotopic PDAC mice treated with rIL33. (FIG. 3g) Tumor volumes of WT and CD103 ⁺ DC-deficient Batf3 ^−/− orthotopic PDAC mice treated with rIL33. (Fig. 3h) Migration of purified DCs to Ccl5. Data were collected at 5 weeks (Fig. 3c,d) and 7 weeks (Fig. 3e,f) after tumor implantation. Data were pooled from two or more independent trials, n≧3/group. n and data points indicate individual mice or (Fig. 3h) individual replicates analyzed separately. P-values were determined by two-tailed log-rank test (Fig. 3a, survival curve), two-way ANOVA (Fig. 3a,b,e,g, tumor volume) and two-tailed Mann-Whitney test (Fig. 3a-f,h). Decided.

Figure 4a-i: PD-1 blockade activates TILC2. (Fig. 4a) Tumor volume and survival, (Fig. 4b) gating, frequency and number, and (Fig. 4c) scRNA-seq (n=7,022) in non-linear representation of the top 15 principal components in treated PDAC mice. ILC2 single cells). Cells are color-coded by cluster (left) or treatment and tissue (right). In (Fig. 4a, b), n = n in the left and right graphs, respectively. (FIG. 4d) Tumor volume of wild-type (WT) or ILC2-deficient PDAC mice treated with αPD-1+rIL33. (Fig. 4e) TILC2 was screen purified from WT or Pdcd1 ^-/- PDAC mice treated with rIL33, transplanted into ILC2-deficient PDAC recipients, and tumor volume was measured. (FIG. 4f-h) TILC2 was sort purified from rIL33-treated PDAC CD45.1 donor mice and transplanted into ILC2-deficient CD45.2 PDAC recipient mice and treated with αPD-1 after cell transplantation. Tumor volume and weight (Fig. 4f), frequency of CD45.1 and CD45.2 cells (Fig. 4g) and frequency of T cells (Fig. 4) in recipient mice 10 weeks after cell transplantation (TILC2s-: all groups; TILC2+: spleen, n=9; DLN, n=7; tumor, n=7). Frequency in FIG. 4g=percentage of immune cells from live donors or recipients. (Fig. 4i) Tumor volume (vehicle, n=13; other groups, n=10) and survival rate (vehicle and αPD-1, n=15; rIL33, n=24) of treated PDAC mice (KPC 52 cells). ; rIL33+PD-1, n=26). DLN, draining lymph node; Data were collected 5 weeks (Fig. 4b), 10 days (Fig. 4c) and 6 weeks (Fig. 4d) after orthotopic tumor cell implantation. Horizontal bars indicate medians and error bars indicate s.e.m. Data are pooled from two or more independent trials with n≧3/group, n and data points indicate individual mice analyzed separately. scRNA-seq data represent purified single cells pooled from biological replicates (vehicle n=10, rIL33 n=5, αPD-1+rIL33 n=5). P-values are calculated by two-way ANOVA with Tukey's multiple comparison post (Fig. 4a, df, i, tumor volume), two-tailed Mann-Whitney (Fig. 4b, d, g, h), and two-tailed log-rank (Fig. 4a, i, survival curve) determined by test. Figure 5a-h: Identification of IL33-dependent ILCs in pancreatic cancer. (Fig. 5a) Gating strategy for identifying human ILCs. The first plot is pre-gated on live cells (DRAQ7 ⁻ ) and singlets. Lineage (Lin) 1 cocktail: CD5, CD11b, CD11c, CD16, FcεR1. Lin2 cocktail: CD3, CD19, TCRα/β. ILCs were identified as Lin ⁻ CD56 ⁻ CD25 ⁺ CD127 ⁺ cells. FMO, fluorescence minus one. (Fig. 5b) Representative images of ILC2 immunofluorescence in tumor tissue microarrays of short- and long-term PDAC survivors (n=96). Arrows, putative ILC2. (Fig. 5c) Top, overall survival in patients with higher (higher) or lower (lower) median intratumoral mRNA levels of ILC-stimulating cytokines. Bottom, Correlation of ILC-activating cytokine expression and immune cytolytic index (CYT) in long-term and short-term survivors of human PDAC. Curves were fitted by linear regression. (Fig. 5d) Gating strategy for identifying mouse ILCs. The first plot is pre-gated on live cells (DRAQ7 ⁻ ) and singlets. Lineage (Lin)1 cocktail: CD5, CD11b, CD11c, FcεR1. Lin2 cocktail: CD3, CD19. ILCs were identified as Lin ⁻ NK1.1 ⁻ CD25 ⁺ CD127 ⁺ and ILC2 as Lin ⁻ NK1.1 ⁻ CD25 ⁺ St2 ⁺ cells. Gating in orthotopic PDAC mice is shown. (Fig. 5e) Intratumoral ILC frequencies in orthotopic PDAC mice established with KPC cell lines 8-1, 18-3, and autologous KPC mice with spontaneous PDAC (KPC ^Spont ). Composite ILC frequencies, such as Fig. 1d, are included for comparison (KPC 4662). (Fig. 5f) ILC phenotype in PDAC mice. Gray curve, isotype control; numbers, mean fluorescence intensity. (Fig. 5g) Proliferation rate of ILCs in tissues of PDAC mice. (FIG. 5h) Changes in non-ILC cell frequency in Rag2 ^−/− PDAC mice treated with αCD90.2 or isotype antibody. Data were analyzed at 14 days (Fig. 5d-f), 10 days (Fig. 5h) or at the indicated time points after tumor implantation. n indicates individual mice analyzed separately in at least two independent studies with n≧2/group. P-values were determined by two-tailed log-rank (Fig. 5c, top), linear regression (Fig. 5c, bottom) or two-tailed Mann-Whitney test (Fig. 5g). P-values in Fig. 5g show the comparison of tumors to all other organs.

Figure 6a-o: Host-derived IL33 activates pancreatic ILC2. (Fig. 6a) ^ILC1- (IL12, IL15, IL18), ^ILC2- (IL25, IL33, TSLP) in orthotopic PDAC tumors (left) and autologous PDAC tumors in KPC mice (right) from a previously reported mRNA microarray. , the mRNA expression of ILC3-induced cytokine (IL23) and IL33 receptor (ST2) were calculated. (FIG. 6b) Representative IL33 immunohistochemistry (IHC) of IL33 ^Low and IL33 ^High human (tissue microarray, n=96) and mouse PDAC (n=3/group). (Fig. 6c) Frequency of human PDAC patients positive for IL33 by IHC on human PDAC tumor microarrays. (Fig. 6d) Multiplex immunofluorescence of IL33, tubular adenocarcinoma marker CK19, myeloid cell carcinoma markers CD11b and Iba in mouse PDAC (top row). Arrows, IL33-expressing cells. Nonimmune (CD45 ⁻ ), immune (CD45 ⁺ ), macrophage (TAM), and monocytic and granulocytic myeloid-derived suppressor cell (M-MDSC and G-MDSC) populations in tumors of IL33 ^Cit reporter PDAC mice. IL33 Mean Fluorescence Intensity (MFI) (lower row). (FIG. 6e) Representative IL33 protein expression by IHC in orthotopic PDAC tumors of Il33 ^+/+ (WT) mice and non-tumor-bearing pancreas of Il33 ^−/− mice (n=3/group). (Fig. 6f) ILC frequencies (top) and cell numbers (bottom) in organs and draining lymph nodes (DLNs) of Il33 ^+/+ and Il33 ^−/− orthotopic PDAC mice. (FIG. 6g) Gating and frequency of IL4 and IL5 expression in intratumoral ILCs of Il33 ^+/+ and Il33 ^−/− orthotopic PDAC mice. (FIG. 6h) ILC2 and immune cell frequencies in orthotopic Rag2 ^−/− and Rag2 ^−/− γc − ^/− PDAC mice treated or not with recombinant IL33 (rIL33) (FIG. 6i). (Fig. 6j) Frequency of ST2 ⁺ tumor ILCs in mice with subcutaneous (SQ) and orthotopic PDAC. (Fig. 6k) Tumors in orthotopic and subcutaneous PDAC mice. (FIG. 6l) Tumor weights in Il33 ^+/+ and Il33 ^−/− littermate PDAC mice. (Fig. 6m) Testing scheme of bone marrow chimeras to assess the contribution of hematopoietic cell-derived IL33 to tumor control. (Fig. 6n) Hematopoietic cell reconstitution and tumor weight in irradiated CD45.1 congenic mice reconstituted with either CD45.2 Il33 ^+/+ or CD45.2 Il33 ^-/- bone marrow (Fig. 6o). Data were collected 14 days (Fig. 6a, d, f, g, j, o) and 10 days (Fig. 6h, i) after tumor implantation. n indicates individual mice analyzed separately in at least two independent studies, n≧2/group. P-values were determined by one-way ANOVA (Fig. 6a) or two-tailed Mann-Whitney test (Fig. 6d, fh, j, l, o). Figure 7a-e: Host-derived IL33 activates pancreatic T-cell immunity. (FIG. 7a) Geneset enrichment analysis of bulk RNA-seq from purified CD45 ⁺ immune cells from Il33 ^+/+ and Il33 ^−/− PDAC mice. Enrichment plots and enrichment scores for three gene sets comparing expression in Il33 ^−/− versus expression in Il33 ^+/+ are shown (n=3 mice/group). FDR, false positive rate. (Fig. 7b) Gating of CD8 ⁺ T cells in Il33 ^+/+ and Il33 ^−/− orthotopic PDAC mice and (Fig. 7c) frequencies of various immune cell types (left) and frequencies of CD4 ⁺ T cell lineages (left). right). (FIG. 7d) T cell central memory (T _cm ) cells in tumor-draining lymph nodes and non-tumor-draining distant lymphoid organs (inguinal lymph nodes and spleen) in Il33 ^+/+ and Il33 ^−/− orthotopic PDAC mice ( CD45 ⁺ CD3 ⁺ CD8 ⁺ CD44 ⁺ CD62L ⁺ ) frequency. (Fig. 7e) Frequency of CD8 ⁺ T cells in subcutaneous PDAC tumors. DC, dendritic cells; MDSC, myeloid-derived suppressor cells; NK, natural killer cells; NKT, natural killer T cells; T _reg , regulatory T cells. Data were analyzed 14 days after tumor implantation or at the indicated time points. Horizontal bars indicate medians, error bars indicate s.e.m. n indicates individual mice analyzed separately in at least two independent studies, n≧2/group. P-values were determined by one-way ANOVA (Fig. 7d). Figure 8a-g: IL33 and ILC do not directly induce tumor cell death. (FIG. 8a) Tumor weights upon administration of vehicle or recombinant murine IL33 (rIL33) to Rag2 ^−/− and Rag2 ^−/− γc − ^/− PDAC mice. (Fig. 8b) Histological tumor cell differentiation status (right) and representative hematoxylin- and eosin-stained sections (left) in Il33 ^+/+ and Il33 ^-/- PDAC mice. (FIG. 8c) Trichrome staining in tumors of Il33 ^+/+ and Il33 ^−/− PDAC mice (n=3/group). (Fig. 8d) Immunohistochemistry of smooth muscle actin in tumors of Il33 ^+/+ and Il33 ^-/- PDAC mice (n=3/group). (FIG. 8e) Intratumoral ST2 expression on KPC cells of Il33 ^+/+ and Il33 ^−/− orthotopic PDAC mice. (Fig. 8f) ST2 expression on live KPC cells after rIL33 treatment in vitro (DRAQ7 stains dead cells) (n=3/group). (Fig. 8g) KPC cell number, viability, proliferation (Ki-67) and apoptosis (annexin) after rIL33 treatment in vitro (n=6/group). n in Figures 8a-e indicates individual mice analyzed separately in at least two independent trials, n > 3/group. Fig. 8f,g n show technical replicates and are representative of at least two independent experiments. P-values were determined by two-tailed Mann-Whitney test (Fig. 8a).

Figure 9a-d: ILC2 induces priming of antigen-specific CD8 ⁺ T cells. (FIG. 9a) Gating of intratumoral ILC2 in ILC2-inactive mice (diphtheria toxin [DT]-treated Icos ^+/+ ; CD4 ^Cre/+ ) and ILC2-deficient mice (DT-treated Icosfl. ^DTR/+ ; CD4 ^Cre/+ ). ting and frequency. (Fig. 9b) Gating and frequency of OVA-specific CD8 ⁺ T cells in the spleens of ILC-intact and ILC-deficient mice. OVA-specific T cells were detected as SIINFEKL (SEQ ID NO: 15)-tetramer ⁺ cells. (Fig. 9c) Gating and frequency of central memory CD8 ⁺ T (TCM) cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ CD44 ⁺ CD62L ⁺ ) in tumor-draining lymph nodes and spleens of ILC intact and ILC-deficient mice. (Fig. 9d) ST2 expression on CD45 ⁺ CD3 ⁺ CD8 ⁺ T cells after tumor implantation in PDAC mice. Data were collected 14 days after tumor implantation or at the indicated time points. DLN, draining lymph node; MFI, mean fluorescence intensity. Horizontal bars indicate medians, error bars indicate s.e.m. n indicates individual mice analyzed separately in at least two independent studies with n≧2/group. P-values were determined by two-tailed Mann-Whitney test (Fig. 9a-c) and two-way ANOVA with Tukey's post-multiple comparison test (Fig. 9d, showing comparison with all other groups of tumor ILCs). . Figure 10a-i: Immunophenotyping in rIL33-treated PDAC mice. (FIG. 10a) Tumor establishment rate of orthotopic and subcutaneous KPC-OVA PDAC tumors in vehicle (veh) and rIL33 treated mice. (Fig. 10b) Gating (left) and frequency (right) of IL18R1 expression on tumor ILCs in subcutaneous (SQ) and orthotopic PDAC mice. (Fig. 10c) Gating (left) and frequency (right) of splenic ILC2 after rIL33 treatment in orthotopic PDAC mice. (Fig. 10d) Gating (left) and frequency (right) of tumor ILC2 after rIL33 treatment in subcutaneous PDAC mice. (Fig. 10e) Gating (left) and frequency (right) of cytokine and PD-1 expression on tumor CD8 ⁺ T cells after rIL33 treatment in orthotopic PDAC mice. (Fig. 10f) Frequency of immune cells in orthotopic PDAC mice treated with vehicle and rIL33. (Fig. 10g) Gating strategy for identifying CD103 ⁺ dendritic cells. (Fig. 10h) Gating (left; tumor) and frequency (right) of ILC2 in tumors and draining lymph nodes (DLN) of wild-type (WT) or Rora ^fl/fl IL7r ^Cre mice (ILC2-deficient) PDAC mice after rIL33 treatment. ). (FIG. 10i) Gating (left) and frequency (right) of PD-1 ⁺ CD8 ⁺ T cells in tumors of rIL33-treated WT and Batf3 ^−/− mice. Data were collected 6 weeks (Fig. 10a), 5 weeks (Fig. 10b) and 3 weeks (Fig. 10i) after tumor implantation. n indicates individual mice analyzed separately in at least two independent studies, n≧2/group. P-values were determined by two-tailed Mann-Whitney test (Fig. 10a, f, i). Figures 11a-c: Single-cell RNA sequencing of tumor and draining lymph node ILC2 in PDAC mice. (Fig. 11a) Study design for in vivo processing, purification and single cell analysis of ILC2. (Fig. 11b,c) Quality metrics. (FIG. 11b) Scatter plot showing the relationship between the number of unique molecular identifiers (# of UMI) and the number of genes (# of genes) for each cell. (FIG. 11c) Violin plot showing the distribution of the number of genes (left), the number of UMIs (middle), and the percentage of normalized reads from mitochondrial genes (right) in each treatment group (columns) and each tissue (rows). ). Each dot represents a single cell. For each treatment group and organ, data represent purified single cells collected from biological replicates of n=10 (vehicle), n=5 (rIL33) and n=5 (αPD-1+rIL33) PDAC mice. Figure 12a-f: Activated ILC2 from tumors and draining lymph nodes have distinct transcriptional signatures. (Fig. 12a) Single-cell analysis of 1,634 rIL33-activated tumor and draining lymph node (DLN) ILC2 (study design outlined in Fig. 11a). UMAP plots show single cells (dots) in the non-linear representation of the top 15 principal components. (Fig. 12a) Expression of transcription factors (TFs) of ILC2 (Gata3, Id2, Rora) and ILC3 (gene, Rorc, protein, Roryt), (Fig. 12b) expression of ILC2 surface markers, and (Fig. 12c) clusters and organs expression. Expression of ILC-1 TF Tbx21 (T-bet) was undetectable. (Fig. 12d,e) cluster and (Fig. 12e) differentially expressed genes by organ (TILC2 and DLN ILC2). (Fig. 12f) Distribution of Ccl5 expression from ILC2 in tumors and DLNs. A violin plot shows the distribution with circles indicating the minimum, maximum and median values. Each dot in Figures 12a and 12b represents a single cell. For each treatment group and organ, data represent purified single cells pooled from biological replicates of n=5 rIL33-treated PDAC mice. P-values are from a two-tailed pairwise Wilcoxon signed-rank test.

Figure 13a-i: Combined treatment of αPD-1 and rIL33 induces a unique transcriptional profile in tumor ILC2. (FIG. 13a) Expression of co-inhibitory immune checkpoint in tumor ILC2 of vehicle-treated PDAC mice by single-cell RNA sequencing (scRNA-seq). (FIG. 13b) Gating and frequency of PD-1 ⁺ ILC2 in vehicle- and rIL33-treated PDAC mice. DLN, draining lymph node. (Fig. 13c) ILC2 frequency in treated PDAC mice. Corresponding tumor volumes, weights, cell numbers and scRNA-seq are shown in Figures 4a-c. (Fig. 13d) scRNA-seq of ILC2 from treated PDAC mice. Expression of ILC1 (gene, Tbx21; protein, Tbet), ILC2 (Gata3, Id2, Rora) and ILC3 (gene, Rorc; protein, Rorγt) transcription factor (TF) in purified tumor and draining lymph node (DLN) ILC2 ) expression. The corresponding UMAP plots by cluster and treatment are shown in Figure 4c. Top differentially expressed genes by treatment and tissue (Fig. 13e), clusters (Fig. 13f) and expression distribution of selected differentially expressed genes by treatment and tissue (Fig. 13g) (tumor: vehicle n=28, rIL33 n =752, rIL33+PD-1 n=2,635; DLNrIL33 n=882, rIL33+PD-1 n=2,725). (Fig. 13h) UMAP plot of 3,387 single tumor ILC2 in a non-linear representation of the top 15 principal components. (Fig. 13i) Treatment differentially expressed genes in tumor ILC2. Each dot in Figures 13d and 13h represents a single cell. For each treatment group and organ, data represent purified single cells pooled from biological replicates of n=10 (vehicle), n=5 (rIL33) and n=5 (αPD-1+rIL33) PDAC mice. . A violin plot shows a distribution with circles indicating the minimum, maximum, and median values. Horizontal bars in Figures 13b and 13c indicate median values. P-values were determined by two-tailed Mann-Whitney test (Fig. 13b,c) and two-tailed Wilcoxon rank sum test (Fig. 13g). Figure 14a-g: Activated tumor ILC2 express PD-1 and colocalize with PD-1 ⁺ T cells. Orthotopic PDAC mice (C57B1/6 WT, Pdcd1 ^−/− , CD45.1) were administered 500 ng carrier-free recombinant murine IL33 daily for 10 days (study design shown in FIG. 4 e, f). Live CD45 ⁺ , Lineage ⁻ , CD90 ⁺ , CD25 ⁺ , ST2 ⁺ tumor ILC2 (TILC2) was sort purified to 98% purity 10 days after transplantation. IL7r ^Cre/+ Rora ^fl/fl (ILC2-deficient) CD45.2 with orthotopic PDAC tumors by intraperitoneal (i.p.) administration of 5× ^{10 5} TILC2 on days 7 and 14 after tumor implantation. Immediately transferred to mice. Control mice were injected i. p. Received an equal volume of PBS via injection. (Fig. 14a) Representative plots of TILC2 sort purification (top) and purity after fractionation (bottom). (Fig. 14b) Representative plots showing PD-1 expression on sort-purified TILC2 from WT and CD45.1 mice in the study design outlined in Fig. 4e,f. (FIG. 14c) Viability and intratumoral CD8 ⁺ T cell frequencies of orthotopic KPC 4662-GFP and KPC 52 PDAC tumors; bars in c represent median values. (Fig. 14d) Frequency of PD-1 ⁺ ILC2 in human PDAC (left) and correlation with PD-1 ⁺ T cells (right). (Fig. 14e) Linear regression analysis of IL33 and PD-1 mRNA in bulk tumor transcriptomes of short- and long-term PDAC survivors (left) and survival associations of PD-1 ⁺ cells in tumor tissue microarrays of short- and long-term PDAC survivors. (right); high and low are defined as higher or lower than the cohort median. (Fig. 14f) A model implicating the IL33-TILC2 axis to T-cell immunity in pancreatic cancer. (Fig. 14g) Expression distribution of co-stimulatory molecules in untreated tumor ILC2 by single-cell RNA sequencing. Study design shown in FIG. 11a; data represent purified single cells pooled from n=10 (vehicle) biological replicates. Data are representative of purity and PD-1 expression in sorted TILC2 in two independent studies with n≧4/group (FIG. 14a,b). n and data points indicate individual mice and patients analyzed separately. P-values were determined by two-tailed Mann-Whitney (Fig. 14c) and two-tailed log rank (Fig. 14c,e, survival curves) tests and linear regression (Fig. 14d,e).

DETAILED DESCRIPTION While some of the key aspects of the present invention are described in the above Summary of Invention and Examples and Claims of this patent application, this Detailed Description section describes the compositions of the invention and Certain additional descriptions of methods are provided and are intended to be read in conjunction with all other sections of this patent application.

Definitions and Abbreviations As used in this specification and claims, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" can be used interchangeably.

Furthermore, "and/or" is taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" used in phrases such as "A and/or B" is intended to include A and B, A or B, A (alone) and B (alone). Similarly, the term "and/or" used in phrases such as "A, B and/or C" means A, B and C; A, B or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone);

Units, prefixes and symbols are written in the form accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range.

When a numerical value is preceded by "about" or "approximately," it includes that numerical value and ±10% of the numerical value.

References in parentheses or superscripts herein refer to numbered publications listed in the "Reference List" at the end of this specification.

Where an embodiment is described with the phrase "comprising," it includes similar embodiments otherwise described with the terms "consisting of" and/or "consisting essentially of."

As used herein, the abbreviation "IL33" means interleukin-33.
As used herein, the abbreviation "rIL33" means recombinant interleukin-33.
As used herein, the abbreviation "PDAC" means pancreatic ductal adenocarcinoma.
As used herein, the abbreviation "ILC2" refers to group 2 innate lymphoid cells.
The abbreviation "TILC2" as used herein means tumor ILC2. All of the embodiments described herein that refer to ILC2 are also intended to encompass TILC2, and for all methods described herein as including ILC2, ILC2 specifically directed to TILC. It should be noted that alternatives are also contemplated by the present invention.

As used herein, the abbreviation "PDX" means patient-derived xenograft.

As used herein, the abbreviation “PD-1” means programmed death 1, also known as programmed death protein 1 or programmed cell death protein 1.

As used herein, the abbreviation PD-L1 means programmed cell death ligand 1--which is the ligand for PD-1.

As used herein, the abbreviation "IP" or "ip" means intraperitoneal administration. The administration of drugs to mice is generally by the IP route, which is considered to be similar to the administration of drugs to humans by the IV route.

As used herein, the abbreviation "IT" means intratumoral administration. For example, drugs injected directly into a tumor are delivered intratumorally.

The abbreviation "IV" as used herein means intravenous administration.

As used herein, the terms "inhibiting" and "blocking" are interchangeable with the terms "inhibit" or "block" and the terms "inhibitor" or "blocker." used for The terms "inhibit" and "block" mean any detectable and statistically significant reduction in a given biological activity.

As used herein, the term "homologous" means deriving from the same species if the members are genetically related or genetically unrelated but genetically similar. , means originating in or members of the same species. "Allograft" or "allogeneic cell therapy" means the administration of cells obtained from a donor (or cells derived from cells obtained from a donor) to a recipient, where the recipient is a donor is the same species as In aspects of the invention involving administration of allogeneic pancreatic ILC2 cells to a subject, allogeneic cells are obtained from a donor of the same species as the subject (ie, recipient) to which the cells are administered. In some embodiments, allogeneic cells are obtained from a donor that has the same MHC/HLA type as the subject (i.e., recipient) to which the cells are administered, i.e., the donor of cells and the recipient of cells are MHC- or HLA-matched. be. In certain embodiments, the cells (e.g., pancreatic ILC2 cells) are (a) obtained from a donor, and (b) maintained and/or cultured and/or expanded and/or activated (e.g., with IL33) ex vivo/in vitro. and (c) subsequently administered to a subject of the same species as the donor. For example, in one embodiment, pancreatic ILC2 cells are obtained from a donor, activated ex vivo/in vitro with IL33, and then administered to a recipient subject of the same species as the donor. Such methods may be referred to as allografts or transplantation or cell therapy methods, and the donor or cells obtained from the donor may be referred to as allogeneic with respect to the recipient. Similarly, in certain embodiments, ILC2 cells are obtained from a donor, activated with IL-33 ex vivo/in vitro, and then administered to a recipient subject of the same species and same MHC/HLA type as the donor.

As used herein, the term "autologous" means derived from or originating from the same subject (ie, the same individual). "Autologous transplantation" or "autologous cell therapy" means the administration of cells obtained from a donor (or cells derived from cells obtained from a donor) to a recipient, wherein the donor and recipient are the same individual. In certain embodiments, the methods of the invention comprise administering autologous pancreatic ILC2 to a subject, wherein the pancreatic ILC2 is obtained from the same individual/subject to which the pancreatic ILC2 is administered (i.e., donor and recipient are the same individual). In certain embodiments, pancreatic ILC2 is (a) obtained from a subject, (b) maintained and/or cultured and/or expanded and/or activated ex vivo/in vitro, and (c) subsequently administered to the same subject. . For example, in some such embodiments, pancreatic ILC2 is obtained from a subject, activated ex vivo/in vitro with IL33, and then administered to the same subject. Such methods may be referred to as autologous transplantation or transplantation or cell therapy, and the donor or cells obtained from the donor may be referred to as being autologous with respect to the recipient.

As used herein, the term "substantially pure," when used in reference to a cell population, is at least about 50%, preferably at least about 75-80%, more preferably at least about 85-85% of the cells that make up the total cell population. It means a population of cells that has 90%, most preferably at least about 95%, of a particular cell marker characteristic/profile. Thus, a "substantially pure" cell population has less than about 50%, preferably less than about 20-25%, more preferably less than about 10-15%, and most Preferably it contains less than about 5%.

As used herein, the term "sorting" with respect to cells refers to separation of cells based on physical properties or the presence of markers (e.g., sorting using side scatter (SSC) and/or forward scatter (FSC), or fluorescence activity by means of automated cell sorting (FACS), eg sorting with labeled antibodies).

As used herein, the term "isolated" in reference to cells means that at least one other cell type, e.g., a designated cell type, is separated from another cell type with which it coexists in nature, e.g. cell type.

Other abbreviations and definitions are described elsewhere herein or are well known in the art.

Active Agents The methods and compositions provided by the invention include a variety of different active agents, including but not limited to IL33 and PD-1 inhibitors.

Any suitable IL33 molecule can be used in those aspects of the invention that include IL33. In preferred embodiments, the IL33 molecule used is the IL33 molecule from the species to which the IL33 is administered. For example, for administration to humans, human IL33 is used, while for administration to mice, mouse IL33 is used. For example, for administration to mice, recombinant murine IL33 (commercially available from R&D Systems, in carrier-containing or carrier-free form) can be used. UniProtKB/Swiss-Prot.: Mouse IL33 having the amino acid sequence defined in Q8BVZ5.1 can be used. Similarly, for administration to humans, recombinant murine IL33 (commercially available from R&D Systems, in carrier-containing or carrier-free form) can be used. Human IL33 having the amino acid sequence defined in UniProtKB/Swiss-Prot.: O95760.1 can be used. Other examples of suitable human IL33 sequences that can be used include, but are not limited to, those consisting of or comprising SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, IL33 is isolated/purified from an animal (eg, mouse, human) that produces it. In some embodiments, IL33 is recombinantly produced—ie, it is expressed in any suitable expression system from recombinant DNA encoding IL33. In some embodiments, IL33 is synthetically produced. In some embodiments, IL33 is modified to increase or improve its half-life, stability, bioavailability, or to improve any other desired biological property. In some embodiments, IL33 comprises a half-life increasing moiety. Any such variant known in the art can be used so long as the altered IL33 retains the ability to bind to and activate the IL33 receptor on ILC2 cells. Examples of modifications that can be used include, but are not limited to, pegylation, conjugation to Fc immunoglobulin domains (such as SEQ ID NO:9), conjugation to albumin binding domains and hexadecenoic acid modifications. Modifications useful for purification or secretion or production of IL33 may also be used, including expression/purification tags (e.g. His-tag such as SEQ ID NO:3 or SEQ ID NO:4), protease recognition sites (e.g. TEV protease recognition sites), secretory signals (such as SEQ ID NO: 6) and linker sequences (such as SEQ ID NO: 7 or 8), but are not limited to these. Examples of modified forms of human IL33 that can be used include SEQ ID NO: 10 (including His-tag and TEV protease recognition site), SEQ ID NO: 11 (including secretion signal, His-tag and linker) or SEQ ID NO: 12 (secretion signal, IgG4 -Fc sequences and linkers). For all examples herein that refer to IL33, the present invention contemplates and encompasses embodiments in which IL33 consists of or comprises any one of SEQ ID NOs: 1, 2, 10, 11 or 12. .

In those aspects of the invention involving PD-1 inhibitors, any suitable PD-1 inhibitor known in the art can be used. In some embodiments, the PD-1 inhibitor is an antibody. In some embodiments, the PD-1 inhibitor is pembrolizumab (Keytruda, Merck), nivolumab (Opdivo, Bristol-Myers Squibb), cemiplimab (Libtayo, Regeneron), AMP-224 (GlaxoSmithKline), AMP-514 (GlaxoSmithKline) and selected from the group consisting of PDR001 (Novartis);

In those aspects of the invention involving PD-L1 inhibitors, any suitable PD-L1 inhibitor known in the art can be used. In some embodiments, the PD-L1 inhibitor is an antibody. In certain embodiments, the PD-L1 inhibitor is atezolizumab (Tecentriq, Roche Genentech), avelumab (Bavencio, Merck Serono and Pfizer), durvalumab (Imfinzi, AstraZeneca), BMS-936559 (Bristol-Myers Squibb), CK-301 ( Checkpoint Therapeutics).

Many of the aspects of the invention described above and/or elsewhere herein involve the use of PD-1 and/or PD-L1 inhibitors. In all cases, it should be noted that the present invention also includes the same embodiments with the variation that only PD-1 inhibitors are used.

In certain embodiments, the methods of the invention can be practiced using analogues, homologues, variants or derivatives that are equivalents of any of the specific active agents described herein. Such analogs, homologues, variants or derivatives should retain the major functional properties of the particular molecules described herein. For example, in the case of PD-1 and/or PD-L1 inhibitors, any suitable analogues, congeners of such agents, provided that they retain PD-1 and/or PD-L1 inhibitory activity; Mutants or derivatives can be used. In the case of IL33, any suitable analogue, homologue, variant or derivative of such agents can be used, provided that it retains the ability to bind to and activate the IL33 receptor on ILC2 cells. can.

In certain aspects, the present invention provides at least one active agent as described herein together with diluents, buffers, carriers, stabilizing agents, dispersing agents, suspending agents, thickening agents, excipients, Compositions are provided that include one or more other ingredients useful in formulating the composition for delivery to a subject, such as preservatives.

Methods of Treatment The present invention provides various methods of treatment. For example, in certain embodiments, the invention calls for an effective amount of one or more of the active agents described herein (e.g., IL33 and/or PD-1 and/or PD-L1 inhibitors) Methods of treatment are provided, comprising administering to a subject. The invention also provides various methods of activating ILC2 cells in pancreatic tumors (eg, PDAC tumors). These methods also generally include administering an effective amount of one or more of the active agents described herein (eg, IL33 and/or PD-1 and/or PD-L1 inhibitors) to a subject in need thereof. including administering. In some aspects, the present invention also provides various cell therapies. These cell therapies involve administering to a recipient subject with PDAC an effective amount of activated donor pancreatic ILC2 cells obtained from a donor subject and activated ex vivo/in vitro by contact with IL33.

As used herein, the terms “treat,” “treating,” and “treatment” refer to detectable treatment in one or more clinical indicators or symptoms associated with pancreatic cancer (e.g., PDAC). achieving and/or implementing methods of achieving such improvements. For example, such terms include reducing the rate of growth of a pancreatic tumor (or pancreatic tumor cells), halting the growth of a pancreatic tumor (or pancreatic tumor cells), regression of a pancreatic tumor (or pancreatic tumor cells), reducing pancreatic tumor size (e.g., measured in terms of tumor volume or tumor mass), downgrading pancreatic tumors, eliminating pancreatic tumors (or pancreatic tumor cells), recurrence of pancreatic tumors preventing, delaying or slowing (rebound), ameliorating symptoms associated with pancreatic tumors, improving survival from pancreatic tumors, inhibiting or reducing the spread (e.g., metastasis) of pancreatic tumors, etc. including but not limited to. For each embodiment described herein that refers to methods of treatment, methods of achieving any one or more of the specified parameters above are also contemplated. For example, for each embodiment described herein that refers to a method of treating pancreatic cancer (e.g., PDAC), the following methods are also contemplated, contemplated, and within the scope of the invention: (a) a pancreatic tumor (or (b) stopping the growth of pancreatic tumors (or pancreatic tumor cells); (c) causing regression of pancreatic tumors (or pancreatic tumor cells); (d) (e) methods of downgrading a pancreatic tumor; (f) methods of removing a pancreatic tumor (or pancreatic tumor cells); (g) (h) methods of ameliorating symptoms associated with pancreatic tumors; (i) methods of improving survival from pancreatic tumors; and (j) pancreatic tumors. A method of inhibiting or reducing expansion (eg, metastasis).

The term "subject" as used herein includes humans, non-human primates, dogs, cats, rodents (such as rats, mice, guinea pigs), cows, pigs, sheep, goats, horses, etc.--used in animal husbandry. All mammalian species are included, as well as all mammals, including but not limited to animals kept as pets, such as in zoos. In some embodiments, the subject is human.

In certain embodiments, the methods and compositions of the invention can be used to treat any pancreatic tumor in a subject in need thereof (ie, a subject with pancreatic cancer). In preferred embodiments, the methods and compositions are used for pancreatic ductal adenocarcinoma (PDAC) in subjects in need thereof (ie, subjects with PDAC). In certain embodiments, the methods and compositions reduce PD-1/PD-L1 inhibitor-resistant PDAC in a subject in need thereof (i.e., a subject with PD-1 and/or PD-L1 inhibitor-resistant PDAC). Used for treatment.

In some embodiments, the subject has a tumor that is refractory to treatment with other methods and/or compositions. The terms "resistant" and "resistant" as used herein are consistent with their normal usage in the art and consistent with the understanding of those terms by physicians (e.g., oncologists) who treat cancer. used for For example, consistent with the ordinary meaning in the art, a tumor or subject is subject to treatment with a treatment method or an agent (or combination of agents) using that method or that agent (or drug combination). combination) may be considered "resistant" if the subject's tumor (or tumor cells) proliferates and/or progresses and/or metastasizes and/or recurs . In some instances, a tumor may be initially sensitive to treatment with a particular method or agent (or combination of agents), but later become resistant to such treatment.

In certain embodiments, the subject has a recurrent pancreatic tumor (e.g., PDAC) after prior treatment with other compositions or methods including, but not limited to, chemotherapy, radiation therapy or surgical resection, or any combination thereof. have In some embodiments, the subject has a previously untreated pancreatic tumor.

As used herein, the term "effective amount" is sufficient to achieve or contribute toward achieving one or more desired clinical outcomes as described in the discussion of "treatment" above, and/or What is meant is an amount of an active agent or cells described herein sufficient to activate ILC2 cells in pancreatic tumors (eg, PDAC tumors). An appropriate "effective" amount in any individual case may be determined using standard techniques known in the art, such as dose escalation studies, depending on the desired route of administration (e.g., systemic versus intratumoral dosing), desired frequency of dosing, and other factors. In some embodiments, an "effective amount" can be an amount previously found to be effective in clinical trials and/or approved for administration to human subjects. For example, in some embodiments, the effective amount of IL33, PD-1 inhibitor or PD-L1 inhibitor is an amount already shown to be effective in clinical trials and/or already approved for administration to human subjects. It may be the amount that is Additionally, an "effective amount" can be determined by whichever method of co-administration is used. One skilled in the art can readily conduct such dosing studies to determine the appropriate dose to use, for example using the assays described in the Examples of this patent - this is subject (dosing studies administration of the agents described herein to animal subjects routinely used in the pharmaceutical arts to perform

For example, in certain embodiments, doses of active agents of the invention may be calculated based on studies in humans or other mammals conducted to determine the efficacy and/or effective amounts of the active agents. Dosage may be determined by methods known in the art, including the pharmaceutical form of the active agent, the route of administration, whether only one active agent is used or multiple active agents (e.g., the first active agent). When the agent is used in combination with a second active agent, the required dose of such first active agent may be lower) and age, weight or the presence of any medical condition affecting drug metabolism. It may vary depending on factors such as patient characteristics, including:

In the embodiments described herein that refer to specific doses of agents to be administered based on mouse studies, those of skill in the art can, for example, Equivalent doses for human studies can be readily determined based on mouse doses using a variety of dosing studies and calculations.

In certain embodiments, appropriate doses of the various active agents described herein can be determined, for example, by starting with doses shown to be effective in mice in the Examples of this patent application, such as in dose escalation studies. It can be determined by conducting a dose test of the type standard in the art.

In some embodiments, one or more active agents are used at about their maximum tolerated dose, eg, as determined in Phase I clinical trials and/or dose escalation studies. In some embodiments, one or more active agents are used at about 90% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 80% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 70% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 60% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 50% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 40% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 30% of their maximum tolerated dose.

Any suitable method or route of administration can be used to deliver the active agents and/or cells or combinations thereof described herein in practicing the treatment methods described herein. In certain embodiments, systemic administration can be employed, eg, oral or intravenous (IV) administration, or any other suitable method or route of systemic administration known in the art. In some embodiments, intratumoral (IT) administration may be employed. In some embodiments, intraperitoneal (IP) delivery may be employed. For example, the active agents described herein can be administered systemically by injection, by infusion through a catheter, with an implantable drug delivery device, or by any other means known in the art. may be administered topically or topically. Depending on the circumstances, for example whether the active agent or the cell is administered, and in the case of an active agent, depending on the properties of the active agent (e.g. its stability, half-life, etc.) , an appropriate delivery method or route can be selected.

In certain embodiments, the compositions and treatment methods provided herein are used in surgical methods (e.g., for tumor resection), radiotherapeutic methods, treatment with chemotherapeutic agents, treatment with angiogenic agents, tyrosine kinase It can be used with other compositions and treatment methods known to be useful in tumor therapy, including but not limited to treatment with inhibitors or treatment with immune checkpoint inhibitors. Also, in certain embodiments, treatment methods provided herein are used to monitor disease status/progression, such as biopsies and diagnostic methods (e.g., MRI or other imaging modalities). It can be used with the method used.

For example, in some embodiments, the methods described herein may be performed prior to surgical resection of a tumor, eg, to shrink the tumor prior to surgical resection. In other embodiments, the methods described herein may be performed both before and after surgical resection of the tumor.

In certain embodiments, the treatment methods described herein can be used in combination with performing diagnostic tests to determine whether a subject has a tumor that is amenable to therapy. For example, in certain embodiments, prior to initiating treatment, diagnostic assays are performed to determine whether a subject has PDAC and/or IL33, such as ST2, among PD-1 and/or PD-L1 expressing cells. Determine if you have a pancreatic cancer (eg, PDAC) that contains cells that express the receptor.

In the cell therapy-based methods described herein, protocols used for other types of cell therapy for tumors (e.g., other types of autologous cell therapy for tumors) are readily adapted for use with ILC2 cells. be able to. For example, modifications of TIL therapy and CAR-T cell therapy can be used. Each of these methods involves obtaining cells from a donor, manipulating those cells in vitro/ex vivo in some way, and then administering those cells to the recipient. For example, methods of obtaining lymphocytes from a donor, isolating/purifying those cells, culturing/maintaining/expanding those cells in vitro/ex vivo, and administering those cells to a recipient are subject to the art. Variations of such methods, well known in the art, can be used in combination with the ILC2-based cell therapy described herein. Regarding purification of pancreatic ILC2 cells obtained in a subject, this can be performed using standard cell separation/purification methods known in the art, such as FAC-based methods. Some ILC2 markers that can be used in such cell separation/purification methods are described in the Examples herein. Other suitable markers are known in the art.

EXAMPLES The present invention is further illustrated by the following non-limiting "examples" and drawings referenced herein. Superscripted numbers in the examples refer to numbered references in the literature listings herein.

Example 1
Tissue-specific innate lymphoid cell activation enhances PD-1 checkpoint blockade immunotherapy in pancreatic cancer
Overview
Group 2 innate lymphoid cells (ILC2) regulate inflammation and immunity in tissues ¹ . ILC2 is detected in cancers of these tissues ² , but its role in cancer immunity and immunotherapy is unclear. Here, we found that ILC2 infiltrates pancreatic ductal adenocarcinoma (PDAC) and activates tissue-specific tumor immunity. Interleukin-33 (IL33) activates tumor ILC2 (TILC2) and CD8 ⁺ T cells in orthotopic pancreatic tumors, but not in ectopic skin tumors, limiting pancreatic-specific tumor growth. Resting and activated TILC2 express the inhibitory checkpoint receptor PD-1, and PD-1 blockers (αPD-1) further proliferate TILC2 and enhance tumor control. PD-1 blockers acted directly on TILC2 to enhance the effects of anti-tumor immunity and αPD-1 immunotherapy, revealing that activated TILC2 is a novel target of αPD-1. Finally, both PD-1 ⁺ TILC2 and PD-1 ⁺ T cells were confirmed to be present in the majority of human PDAC. From the above, the present inventors revealed that ILC2 is a novel anti-cancer immune cell for PDAC immunotherapy. More broadly, ILC2 emerges as a tissue-specific cancer immune enhancer that amplifies the effects of αPD-1 immunotherapy. Co-targeting anti-cancer ILC2 and T cells may be a broadly applicable immunotherapeutic approach, as ILC2 and T cells are comorbid in human cancers sharing activating and inhibiting pathways. .

TILC2 lacks immune cell lineage markers (Lin ⁻ ), but ILC (CD25, CD127) ¹ , as well as ILC2 (IL33-receptor ST2), in unselected human primary PDAC infiltrating pancreatic cancer . /IL1RL1/IL33R and GATA3) markers were found in tumor cells (Fig. la, Fig. 5a). These putative TILC2s are more pronounced in rare long ^- term PDAC survivors with "hot" tumors (rich in activated CD8 ⁺ T cells) compared to short-term survivors of cold tumors. enriched, and high TILC2 frequency correlated with long survival (Fig. 1b, Fig. 5b). Consistently, high bulk tumor RNA expression of the ILC2-activating cytokine IL33, but not other ILC-activating cytokines, was associated with longer survival (Fig. 1c, Fig. 5c). Furthermore, IL33, but not other ILC-activating cytokines, correlated with high intratumor immune cytolytic activity (Fig. 1c, Fig. 5c). These data, assessing RNA abundance rather than protein abundance, suggested that IL33 and TILC2 activate anti-tumor immunity in human PDAC.

Next, we examined the presence of ILCs in tumors from Kras- and p53-mutated autochthonous "KPC" ^mice4 and orthotopic PDAC mouse models (PDAC ^mice5,6 ). rice field. Both models detected TILC2 with a phenotype similar to that in human PDAC and mouse ILC2 ^1,7 (Fig. 1d, Fig. 5d-f). Murine TILC2 proliferated in tumors but not in adjacent organs (Fig. 1d, Fig. 5g), consistent with its tissue homeostasis8 and targeting the lymphocyte antigen ^CD90.2 in Rag2 ^-/- mice to reduce it. (Fig. 1e, Fig. 5h). Thus, ILC2 is a conserved cell that grows locally in mouse and human PDAC.

In identifying signals that induce proliferation of TILC2, both PDAC and KPC mice ¹¹ showed that IL33 had the highest expression within tumors compared to other ILC-inducing cytokines (Fig. 6a) ⁶ , human and mouse PDAC. (FIGS. 6b-c), with maximal expression in intratumoral myeloid cells ¹² , 13 (FIGS. 6d, e). To understand the role of IL33 and TILC2 in PDAC immunity, we examined TILC2 dependence in IL33 ^High PDAC mice, which reflects IL33 ^High , ILC2-enriched inflammatory tumors in human PDAC long-term survivors. IL33 ^−/− PDAC mice had reduced TILC2 frequencies, numbers (FIGS. 1f, 6f) and cytokine production (FIG. 6g) compared to IL33 ^+/+ PDAC mice, suggesting that TILC2 proliferation and function It was found to be IL33 dependent. Consistently, recombinant IL33 (rIL33) proliferated ILCs in ILC-proficient Rag2 ^−/− PDAC mice, but not in ILC-deficient Rag2 ^−/− γc − ^/− PDAC mice. was not allowed (Fig. 6h,i). These studies showed that IL33 expanded PDAC TILC2.

TILC2 enhances tumor immunity in tissues Because ^ILC2 has a tissue-specific phenotype, we hypothesized that the effect of TILC2 on PDAC immunity was tissue-specific. . To test this, we contrasted the effect of IL33 deletion on pancreatic and skin tumor growth (pancreatic TILC2 expresses ST2, skin TILC2 does not; FIG. 6j ^14,15 ). Compared to Il33 ^+/+ animals, Il33 ^−/− mice with orthotopic PDAC had larger tumors and accelerated tumor growth, in contrast to subcutaneous PDAC mice, which do not display an IL33-dependent phenotype. , had poor survival (Fig. 6a) (Fig. 2b, Fig. 6k). Although these mice are fully backcrossed to identical genetic backgrounds, the observation of larger tumors in IL33 ^−/− vs. IL33 ^+/+ littermates suggests that these differences may represent a potential small genetic background. We confirmed that it was not caused by a mismatch (Fig. 6l). These anti-tumor effects were dependent on host hematopoietic cell-derived IL33, as chimeric mice engrafted with Il33 ^−/− bone marrow had larger tumors than control mice (FIG. 6m-o). RNA-sequencing (RNA-seq) of purified CD45 ⁺ intratumoral immune cells from Il33 ^+/+ and Il33 ^−/− orthotopic PDAC mice showed that Il33 ^−/− PDAC immune cells were associated with T cell activation and MHC-I Transcriptional signals for antigen processing were reduced, suggesting a deficiency in T cell priming (Fig. 7a). Consistently, Il33 ^−/− orthotopic PDAC mice had lower frequencies of tumor-infiltrating CD8 ⁺ T cells, but not subcutaneous, with no consistent change in frequencies of other immune cells, draining lymph node (DLN) Central memory CD8 ⁺ T cells (TCMs) were decreased in the cells, but not in distant lymph nodes (Fig. 2c, Fig. 7c-e). The increased tumor size of IL33 ^−/− mice compared with Il33 ^+/+ mice was abolished by pan-T cell depletion (FIG. 2d), and there was no difference in tumor weight of rIL33-treated Rag2 ^−/− PDAC mice. (Fig. 8a), confirming that the anti-tumor effect of IL33 is T-cell mediated. Orthotopic tumors of Il33 ^−/− and Il33 ^+/+ PDAC mice also had similar histology, collagen and fibroblast content (FIGS. 8b–d), demonstrating that rIL33 directed to tumor cells in vitro. There was no effect (Fig. 8e-g), indicating that IL33 does not directly affect tumor or stromal cells. These data revealed that IL33 activates tissue-specific cancer immunity by activating TILC2 and inducing CD8 ⁺ T cells.

We next investigated whether the effect of IL33 on CD8 ⁺ T cells was tissue-specific by examining the rejection phenotype of KPC cells expressing the CD8 ⁺ T cell rejection antigen ovalbumin (KPC-OVA). It was examined by contrasting different tissue sites. Interestingly, 70% of Il33 ^+/+ mice rejected orthotopic KPC-OVA tumors, whereas 0% of Il33 ^−/− mice. On the other hand, 100% of Il33 ^+/+ and Il33 ^−/− mice rejected subcutaneous KPC-OVA tumors (FIG. 2e). To assess whether this phenotype is due to ILC2 deletion and ineffective priming of CD8 ⁺ T cells, iCOS-1, which allows ILC2 depletion by diphtheria toxin while sparing ICOS ⁺ CD4 ⁺ T ^cells16 Acute depletion and testing of antigen-specific CD8 ⁺ T cells in the DLN were performed using T mice (Fig. 2f, Fig. 9a). ILC2 depletion recapitulated the Il33 ^−/− phenotype, with higher tumor rejection rates in orthotopic KPC-OVA tumors, larger tumor sizes, and no difference in subcutaneous tumors (Fig. 2f), but decreased rejection assessment time and Given the difference in depletion effects, this was the expected variation compared to Il33 ^−/− mice. Tetramer analysis in ILC2-depleted orthotopic KPC-OVA mice showed reduced OVA-specific CD8 ⁺ T cells in the DLN and spleen, and reduced CD8 ⁺ TCM in the DLN (as seen in IL33 ^−/− mice). (Fig. 2g, Fig. 9B, C). Therefore, ILC2 deletion partially expresses IL33 deletion. Although a direct effect of IL33 on CD8 ⁺ T cells cannot be denied, it was found that ST2 was not expressed in intratumoral CD8 ⁺ T cells (Fig. 9d). Collectively, these loss-of-function studies suggested that the IL33-TILC2 axis primes tissue-specific CD8+ T cell PDAC immunity.

Next, to investigate whether rIL33 treatment has similar tissue-specific anti-tumor effects, we demonstrated that rIL33 blocked tumor formation, prolonged survival, and subcutaneously in orthotopic PDAC mice. We found that PDAC mice had no effect, resulting in progressive tumor growth and ulceration requiring euthanasia (Fig. 3a), and had similar tissue-specific anti-tumor effects in KPC-OVA PDAC mice (Fig. 10a). ). Similarly, rIL18, a cytokine that preferentially activates ^{IL18R +} cutaneous ILC2s, restricted proliferation of subcutaneous PDAC infiltrated with IL18R ⁺ ILC, but not orthotopic PDAC without IL18R ⁺ ILC. (Fig. 3b, Fig. 10b). rIL33 selectively expanded ILC2 in the DLN and tumors of orthotopic PDAC mice (Fig. 3c), with no changes in splenic or subcutaneous PDAC (Fig. 10c,d). Expansion of ILC2 was accompanied by enhanced intratumoral CD8 ⁺ T cell cytokine capacity and upregulation of PD-1 (Fig. 10e), while no consistent changes were seen in other intratumoral immune cells (Fig. 10f). , the possibility of regulating their functions cannot be ruled out. Consistent with ILC2 indirectly priming anti-tumor CD8 ⁺ T cells, rIL33 treatment doubled intratumoral CD103 ⁺ dendritic cells (DCs) (Fig. 3d, Fig. 10g), increasing the number of CD8 ⁺ T cells. Primed and recruited to ^PDAC6 . To investigate whether the effects of rIL33 are dependent on ILC2, we administered rIL33 to Rora ^fl/fl Il7r ^Cre/+ mice with PDAC to constitutively delete ILC2 ¹⁶ . ILC2 deletion (Fig. 10h) compromised rIL33 efficacy (Fig. 3e) and attenuated the increase in CD103 ⁺ DCs within the tumor (Fig. 3f). rIL33 showed no anti-tumor effect in CD103 ⁺ DC-deficient Batf3 ^−/− mice (FIG. 3g), nor could it induce the expression of intratumoral CD8 ⁺ T cells PD-1 (FIG. 10i), indicating tumor control by rIL33. could be demonstrated to be essential for CD103 ⁺ DC. To confirm whether TILC2 produces chemokines and recruits DCs to tumors, we used single-cell RNA-seq (scRNA-seq) (FIGS. 11a-c) and found that activating TILC2 and DLN ILC2 It was found to retain markers of identity but display distinct transcriptional profiles (Fig. 12a-e), rIL33-activated TILC2 encodes a chemokine that recruits CD103 ⁺ DCs to tumors. We selectively expressed Ccl5 (Fig. 12f) ¹⁷ and found to induce efficient DC migration in vitro (Fig. 3h). Taken together, these data suggest that rIL33 expands TILC2, potentially recruits CD103 ⁺ DCs to tumors via Ccl5 production, and activates CD8 ⁺ T cells to induce therapeutic tumor immunity. did.

Activation of TILC2 by PD-1 Blockers Since stimulation of ILC2 with rIL33 has an anti-tumor effect, the present inventors sought strategies to further enhance ILC2 activation. Recent data indicate that, like T cells, ILC2 also regulates its activity through a co-inhibitor ^2,18 immune checkpoint pathway. Specifically, the immune checkpoint PD-1 regulates the development of murine ILC2 ¹⁹ , marks effector ILCs ¹⁹ , and when genetically deleted or inhibited with a blocking antibody (PD-1), IL33 activation increases. ILC2 becomes more proliferative and effector function in mice and humans ²⁰ . PD-1 ⁺ ILC2 is also present in human tumors ² . However, simultaneous activation and inhibition of ILC2 for cancer therapy is a relatively unsolved area.

Using scRNA-seq (FIGS. 11a-c), we found that PD-1 was the only detectable co-inhibitory molecule expressed by TILC2 at baseline (FIG. 13a). rIL33 treatment upregulated PD-1 in a subset of TILC2, but not in DLN ILC2 (Fig. 13b), suggesting that PD-1 may functionally repress activated TILC2. It was suggested. Therefore, the present inventors investigated whether rIL33 and αPD-1 could be combined to cooperatively activate TILC2 and enhance the antitumor effect. Consistent with PD-1 being expressed only on rIL33-activated TILC2, αPD-1 alone induced a partial response as previously reported for PDAC ₆ (Fig. 4a), No significant change in TILC2 frequency was observed (Fig. 4b, Fig. 13c). Combining rIL33 with αPD-1 maximally increased ILC2 in tumors and DLNs (Fig. 4b) and enhanced tumor control compared to PD-1 alone (Fig. 4a). To investigate whether αPD-1 is activating ILC2 by blocking endogenous PD-1, we compared the single-cell transcriptional profiles of TILC2 and DLN ILC2 after in vivo treatment. Although TILC2 retained the transcriptional and cellular identity of ILC2 regardless of treatment (Fig. 13d), TILC2 in rIL33- and αPD-1-treated PDAC mice displayed a unique transcriptional phenotype compared to all other conditions. (Fig. 4c), containing ILC2-specific markers, canonical (amphiregulin [Areg]) ¹⁴ and non-canonical (CXCL2) ²¹ effector molecules, cell activation mechanisms (Junb, Fosl2, Ybx1), increased expression of co-inhibitory immune checkpoints was seen (FIG. 13e-i). Finally, the anti-tumor effect of the dual treatment was abolished in ILC2-deficient mice (Fig. 4d), demonstrating that ILC2 is required for the efficacy of the αPD-1 and rIL33 dual treatment. These results suggested that αPD-1 preferentially amplifies activated TILC2 not only on T cells, but probably by inhibiting the PD-1 pathway on ILC2.

PD-1 TILC2 inhibition is cellular endogenous To determine whether blockade of the intracellular PD-1 pathway in activated TILC2 contributes to the antitumor effects of dual therapy, we screened Purified rIL33-activated PD-1-proficient (wild-type [WT]) or PD-1-deficient (Pdcd1 ^−/− ) TILC2 were transplanted into tumor-bearing ILC2-deficient mice (FIG. 4e, FIG. 14a). Although WT TILC2 delivery had no anti-tumor effect in established tumors, Pdcd1 ^−/− TILC2 limits tumor growth, indicating that disrupting PD-1 signaling on TILC2 can enhance tumor control (Fig. 4e). We next examined whether rIL33-activated PD-1 ⁺ TILC2 could directly amplify the effects of PD-1 therapy in established tumors. We transplanted the screen-purified rIL33-activated congenic line CD45.1 ⁺ TILC2 into tumor-established CD45.2 ⁺ ILC2-deficient mice and treated with PD-1 after transplantation (Fig. 4f). Transplanted TILC2 was >97% PD-1 ⁺ (Fig. 10b) and accumulated in tumors and DLNs of αPD-1-treated recipient mice, but not in the spleen, persisting up to 9 weeks post-transplantation. (Fig. 4g). Transplantation of PD-1 ⁺ TILC2 enhanced the potency of αPD-1, limited tumor growth, and increased T cell frequencies in tumors and DLNs (not including spleen) of recipient mice (Fig. 4h). . Together these data indicated that blockade of PD-1 signaling in rIL33-activated TILC2 had a direct anti-tumor effect and amplified PD-1 effects.

To examine rIL33 and PD-1 efficacy in IL33 ^Low αPD-1-resistant tumors, IL33 ^Low tumors (Fig. 6b) were generated to mimic the immunological and survival characteristics of IL33 ^Low short-term human PDAC survivors. , an aggressive "non-inflammatory" PDAC model (KPC52) with 50% fewer CD8 ⁺ T cells and a median survival of only 2 weeks (Fig. 14c). KPC52 PDAC mice do not show the sequential stages of PDAC tumorigenesis from preinvasive neoplasm to invasive PDAC as seen in spontaneous KPC mice, but αPD seen in spontaneous KPC mice and human PDAC. -1 resistance is reproduced (Fig. 4i). We found that the combination of rIL33 and αPD-1 reduced tumor volume by nearly 40% and improved mouse survival by nearly 50% (Fig. 4i). Finally, to assess the potential treatment of PDAC patients with combination therapy of rIL33 and αPD-1, we found that nearly 60% of human PDAC have low frequencies of PD-1 ⁺ TILC2 and PD-1 ⁺ T cells, suggesting that the 2 There was a significant correlation between the two cell types (Fig. 14d), suggesting that they are frequently co-occurring in human PDAC. Furthermore, IL33 mRNA was significantly correlated with PD-1 mRNA, which is associated with prolonged survival (Fig. 14e), suggesting that the IL33-PD-1 axis may positively affect human PDAC survival. suggested ²² . Taken together, activation of ILC2 with rIL33 can amplify responses to αPD-1 in both PD-1 partially sensitive and PD-1 resistant tumors.

Discussion Our results suggest that activating ILC2 may be a broader strategy to promote T cell priming in ILC2-invasive cancers (Fig. 14f). However, given the tissue-specific phenotype of ILC2, further studies are needed whether activating them would have similar effects in various cancer types.

Although ILC2 expresses an immune checkpoint, it was unclear whether immune checkpoint inhibition therapy could exert its ability. We confirm that blocking PD-1 on activated ILC2 has anti-tumor effects and suggest that ILC2 partially contributes to the effects of PD-1 pathway blockade in human cancers. more broadly emphasizes that differences in checkpoint block responses can depend on tissue-specific factors.

Expression of several immunoregulatory molecules by activated ILC2 (Fig. 14g) suggests that a wider range of checkpoints may be targeted in tumor ILC2 and T cells. Because ILC2 and T cells have common co-stimulatory and co-inhibitory pathways and coexist in human cancers, strategies to collectively target ILC2 and T cells for cancer immunotherapy are needed.

The data presented in this example were published by the inventors in the journal Nature (Moral et al., “ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity” Nature, 2020, Mar, Vol. 579(7797), pp 130-135.doi: 10.1038/s41586-020-2015-4, Epub 2020 Feb 19). The entire contents of this document, including supplementary material, are incorporated herein by reference.

Example 2
material and method
This example describes the materials and methods used in conducting the experiments described in Example 1.
mouse
C57BL/6 (wild type, WT, CD45.2), C57BL/6 CD45.1, Rag2 ^−/− , Rag2 ^−/− c ^−/− , Batf3 ^−/− and Pdcd1 ^−/− mice were from Jackson Labs. Purchased. Il33 ^−/− , Il33 ^Cit/+ are M. J. It was a gift from Rosen. Cd4 ^Cre/+ Icos ^fl-Dtr/+ and Il7r ^Cre/+ Rorα ^fl/fl were isolated from A. N. J. A gift from McKenzie and previously reported ^16,23 . For all experiments, mice aged 6-12 weeks were age- and sex-matched, randomly assigned to specific treatment groups, and a total of at least two independent experiments were performed. Pdx1-Cre; LSL-Kras ^G12D/+ ; LSL-Trp53 ^R172H/+ (KPC mice) have been previously reported ⁴ . Experimental sample sizes were determined without performing formal power calculations. Animals were bred and maintained in a specific pathogen-free animal facility, and all experiments were performed in accordance with Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee (IACUC)-approved protocols and in compliance with all relevant ethical regulations. implemented in compliance.

Cell Lines and Animal Experiments All tumor cell lines are derived from KPC mice. LSL-Kras ^G12D/+ ; LSL-Trp53 ^R172H/+ (gift of RH Vonderheide) KPC4662 cells were transfected with GFP and used for all experiments unless otherwise indicated. KPC8-1,18-3,52 cells from Ptf1a-Cre; LSL-Kras ^G12D/+ ; LSL-Trp53 ^R172H/+ mice were transformed into C. It was a gift from Iacobuzio-Donahue. KPC4662 cells engineered to express OVA have been previously reported ²⁴ (gift of RH Vonderheide). All cell lines were accepted as bonafide PDAC cell lines based on histopathological verification by an expert pancreatic cancer pathologist. Orthotopic tumors established with KPC4662 cells were IL33 ^High and transiently decreased in size due to PD-1 therapy initiated at the time of transplantation (PD-1 partial sensitivity). Orthotopic tumors established with KPC52 cells were IL33 ^Low and did not decrease in size with PD-1 therapy initiated at the time of transplantation (PD-1 resistance). All cell lines were routinely tested using the MycoAlert Mycoplasma Detection Kit (Lonza). Orthotopic PDAC tumors were established as previously reported ⁵ . Briefly, mice were anesthetized with a ketamine/xylazine cocktail and a small (7 mm) left lateral flank incision was made. Tumor cells (10 ⁶ KPC cells/mouse; 1.25×10 ⁵ KPC-OVA cells/mouse) were suspended in Matrigel (Becton Dickinson) and diluted 1:1 with cold phosphate-buffered saline (PBS) ( A total volume of 50 μl) was injected into the pancreas tail with a 26 gauge needle. Successful injection was confirmed by the appearance of a bubble without intraperitoneal leakage. The abdominal wall was closed with absorbable Vicryl RAPIDE sutures (Ethicon) and the skin with wound clips (Roboz). For subcutaneous PDAC tumors, tumor cells (10 ⁶ KPC cells/mouse; 1.25×10 ⁵ KPC-OVA cells/mouse) were resuspended in sterile PBS (Fisher Scientific) and implanted subcutaneously. Mice were sacrificed at the indicated time points and processed for tissue examination or flow cytometry. Autochthonous KPC mice were sacrificed when tumors were detectable by ultrasound. Tumor volumes were measured on orthotopic tumors using continuous ultrasound (Vevo 2100 Linear Array Imaging and Vivo LAB Version 3.1.1, Fuji Film Visual Sonics) as previously reported ²⁵ . For subcutaneous tumors, tumor length and width were measured with vernier calipers every 2-3 days, and tumor volume was calculated as Volume=1/ ² length×width2. For survival analysis, survival was determined by the health status of mice requiring tumor volume greater than 500 mm ³ or requiring euthanasia as defined by institutional IACUC guidelines. None of the mouse tumors exceeded an IACUC-defined maximum tumor volume of 2 cm ³ . No randomization was performed during the handling of the experimental mice as the treatment groups needed to be known.

T-cell depleting anti-mouse CD4 antibody (clone GK1.5, BioXcell, InVivoPlus) 250 μg and anti-mouse CD8a antibody (clone 2.43, BioXcell, InVivoPlus) 250 μg were injected intraperitoneally (ip) to deplete CD4 and CD8 cells. exhausted. Control mice received a rat IgG2b isotype control (clone LTF-2, BioXcell, InVivoPlus). Mice were treated daily starting 3 days prior to tumor implantation and then every 3 days for the duration of the experiment. Depletion of CD4 ⁺ and CD8 ⁺ T cells was confirmed by flow cytometric analysis of tumors and secondary lymphoid organs (>85% depletion).

ILCs in ILC-depleted Rag2 ^−/− mice were induced by anti-mouse CD90.2 (clonal 30-H12, BioXCell) 300 μg i. p. injected and decreased ²⁶ . Cd4 ^Cre/+ Icos ^fl-DTR/+ experimental mice and Cd4 ^Cre/+ Icos ^+/+ control mice were injected ip with diphtheria toxin (Sigma Aldrich) at a dose of 25 ng/g mouse body weight. p. Injected and treated to deplete ILC2. Mice were treated the day before tumor implantation and then given every other day for a total of 5 doses as previously reported ¹⁶ . ILC2 depletion was confirmed by flow cytometric analysis of tumors (Fig. 9a).

Bone marrow chimeric bone marrow was harvested from CD45.2 congenital labeled donor mice, filtered through a 70 mm filter, centrifuged and resuspended in sterile PBS to a concentration of 10 ⁸ viable cells per 200 μl. CD45.1 congenitally labeled C57BL/6J recipient mice were irradiated (5.5 Gy x 2, 6 hour intervals) 24 hours prior to bone marrow transplantation and maintained on endofloxacin water for 4 weeks post-irradiation. A single cell suspension of CD45.2 bone marrow chimeras in sterile PBS (10 ⁸ viable cells per recipient mouse) was implanted into each recipient mouse by retro-orbital injection. Reconstitution was confirmed by flow cytometry of peripheral blood 4 and 8 weeks after transplantation. Tumor implantation experiments were performed 12 weeks after implantation.

For recombinant IL33, IL18 and PD-1 blocking rIL33, mice were injected intraperitoneally with 500 ng of carrier-free recombinant mouse IL33 (R&D Systems) in sterile PBS daily for 7 days and then every 2 days as previously reported. ¹⁵ were treated as For rIL18, 2 μg of carrier-free recombinant mouse IL-18 (R&D Systems) was administered i. p. Mice were treated with injections ²⁷ . The chimeric anti-mouse PD-1 antibody (4H2) used in this study, designed as a mouse IgG1 isotype monoclonal antibody (mAb), binds to PD-1-expressing CHO transfectants and induces activation of these cells. It was shown to block the binding of PD-L1 and PD-L2. The affinity of 4H2 for mouse PD-1 measured by surface plasmon resonance using PD-1-Fc was 4.68×10 ⁻⁹ M. Each batch is certified to be <0.5 EU/mg endotoxin and >95% pure. All dosing solutions were prepared in PBS. Mice were injected i.p. with 250 μg of anti-PD1 antibody every 2 days. p. treated by injection. A partial response was defined as a transient reduction in tumor size during continued administration of αPD-1 followed by re-growth. Resistant was defined as no reduction in tumor size during continued administration of αPD-1.

Tissues for all human samples were collected at MSKCC after approval of the study protocol by the MSKCC Institutional Review Board. Informed consent was obtained for all patients. This study was conducted in strict compliance with all institutional ethical codes. All tumor samples were surgically resected primary PDAC.

tissue microarray. Tissue microarrays (TMA) were analyzed from tumor and adjacent non-formalin-fixed, paraffin-embedded tissue blocks of short-term (n=45 tumors, 5 normal tissues) and long-term survivors (n=51 tumors, 5 normal tissues) of PDAC. It was constructed from tumor cores as previously reported ³ . Patient subsets were randomly selected for tissue microarray construction. Patients receiving neoadjuvant therapy were excluded. All tumors underwent pathological review and histological confirmation by two PDAC specialist pathologists prior to analysis. To exclude perioperative mortality, long-term survivors were defined as patients with overall survival >3 years from surgery, and short-term survivors as patients with survival >3 months to <1 year from surgery. ILC2 ^High and ILC2 ^Low were defined as greater than or less than the median ILC2 frequency across the TMA cohort, respectively.

Tumor transcriptome profiling. Patient subsets were randomly selected and transcriptome profiling was performed as previously described ³ . Patients within the TMA cohort with tumor tissue available for transcriptome evaluation were included in the analysis of Figure 1b so that RNA expression could be confirmed with protein. Extracted RNA was qualified on an Agilent BioAnalyzer and quantified by fluorometry (Ribogreen). RNA preparation for whole transcriptome expression analysis was performed using the WT Pico Reagent kit (Affymetrix). To capture both coding and multiple non-coding RNAs, reverse transcription was initiated at the polyA tail as well as the full length of the RNA. RNA amplification was achieved by linear amplification using low cycle PCR and T7 in vitro transcription technology. The cRNA was then converted into a biotinylated sense strand DNA hybridization target. Prepared targets were hybridized to GeneChip Human Transcriptome Array 2.0 (Affymetrix). Washing was performed using the GeneChip Hybridization, Wash and Stain kit using Fluidics Station 450/250. Scanning of arrays was performed with a GeneChip Scanner 3000. Data analysis of arrays was performed using Affymetrix Expression Console software (SST-RMA algorithm to summarize the signal from array probesets). Immune cytolytic activity was determined as previously reported ²⁸ .

Cell Isolation Murine PDAC tumors and human PDAC tumors and adjacent pancreases were mechanically dissociated and treated with collagenase (collagenase II for mouse tumors and collagenase IV for human tumors, both 5 mg/ml; Worthington Biochemical Corp., Fisher Scientific), Incubated for 30 minutes at 37° C. in DNAse I (0.5 mg/ml; Roche Diagnostics) and Hank's balanced salt solution (Gibco, Fisher Scientific). Digestion was then stopped with fetal bovine serum (FBS, Life Technologies) and the cells were filtered sequentially through 100 mm and 40 mm nylon cell strainers (Falcon, Fisher Scientific). Tumors, adjacent pancreas and lymph nodes were then mechanically separated and filtered through 100 mm and 40 mm nylon cell strainers (Falcon, Fisher Scientific) using PBS with 1% FBS (Life Technologies). The spleen was mechanically separated, filtered with 70 mm and 40 mm nylon cell strainers (Falcon, Fisher Scientific) using PBS containing 1% FBS, and then red blood cells were lysed (RBC lysis buffer, ThermoFisher Scientific). Mouse Fc receptors were blocked with an FcεRIII/II specific antibody (1 μg per 1×10 ⁶ cells; clone 2.4G2, Bio X Cell).

ILC2 adoptively transferred CD45.1 C57B1/6 or Pdcd1 ^−/− orthotopic PDAC mice were administered 500 ng of carrier-free recombinant murine IL33 (R&D Systems) in sterile PBS for 10 days. Viable, CD45 ⁺ , lineage ⁻ , CD90 ⁺ , CD25 ⁺ , ST2 ⁺ TILC2 were sort-purified to 98% purity on day 10 post-implantation using the Aria Cell sorter (BD Biosciences). 5×10 ⁵ tumor ILC2 were administered i.p. 7 and 14 days after tumor implantation. p. IL7r ^Cre/+ Ror ^fl/fl CD45.2 mice bearing orthotopic PDAC tumors were immediately implanted by injection. Control mice received the same amount of PBS i. p. Administered by injection. αPD-1 treatment in recipient mice was initiated on the day of ILC2 cell transplantation. Tissues were harvested at the indicated time points.

Flow cytometry Single cell suspensions were stained with an antibody cocktail at 4°C in the dark and after washing were analyzed on a FACS LSR Fortessa (BD Biosciences). Murine ILCs were defined as viable, CD45 ⁺ , lineage ⁻ (CD3, CD5, NK1.1, CD11b, CD11c, CD19, ^FcεR1 ), CD25 ⁺ , CD127 ⁺ cells as previously reported 7,1 . Mouse immune cells were defined as follows: ILC2 = viable, CD45 ⁺ , lineage ⁻ , CD25 ⁺ , ST2 ⁺ cells; central memory T cells = viable, CD45 ⁺ , CD3 ⁺ , NK1.1 ⁻ , CD8. ⁺ , CD62l ⁺ , CD44 ⁺ ; Dendritic cells = viable cells, CD45 ⁺ , CD3 ⁻ , NK1.1 ⁻ , Gr1 ⁻ , F4/80 ⁻ , CD11c ⁺ , MHC-II ⁺ ; B cells = viable cells, CD45 ⁺ CD4 ⁺ T cells = viable, CD45 ⁺ , CD3 ⁺ , CD4 ⁺ ; CD8 ⁺ T cells = viable, CD45 ⁺ , CD3 ⁺ , CD3 ⁻ , CD19 ⁺ ; T cells = viable, CD45 ⁺ , CD3 ⁺ ; CD8 ⁺ ; regulatory T cells = viable cells, CD45 ⁺ , CD3 ⁺ , CD4 ⁺ FoxP3 ⁺ ; tumor-associated macrophages = viable cells, CD45 ⁺ , CD11b ⁺ , F4/80 ⁺ , GR1 ⁻ ; myeloid-derived suppressor cells (MDSC) = live cells, CD45 ⁺ , CD3 ⁻ , CD11b ⁺ , F4/80 ⁻ , GR1 ⁺ ; mouse cells were stained with the following antibodies: Biolegend's CD45 (clone 30-F11, Pacific Blue), CD45.1 (clone A20). NK1.1 (clone PK136, APC), Gr-1 (clone RB6-8C5, BV605), CD103 (clone 2E7, BV711); BD Biosciences, CD5 (clone 53-7.3, APC); CD11c (clone HL3, APC), NK1.1 (clone PK136, BV605), CD4 (clone RM4-5, BV786), CD62L (clone MEL-14, APC), CD19 (clone 1D3, BV510), Ly6C (clone AL -21, PerCP-Cy5.5), Ly6G (clone 1A8, AF700), PD1 (clone J43, BV605), TNF-α (clone MP6-XT22, BV510), IFN-γ (clone XMG1.2, APC-Cy7 ), CD90.2 (clone 53-2.1, BV786), Tbet (clone Q4-46, BV711), Ror-t (clone Q31-378, BV786), Gata3 (clone L50-823, PE-Cy7) and IL4 (clone 11B11, BV650); Th ermoFisher Scientific CD3 (clone 17A2, Alexa Fluor 700), CD11b (clone M1/70, APC), CD11b (clone M1/70, PerCP-Cy5.5), CD8 (clone 53-6.7, Alexa Fluor 700) , CD19 (clone 1D3, Alexa Fluor 700), FcR1 (clone MAR-1, APC), F4/80 (clone BM8, PE-Cy5), CD3 (clone 145-2C11, PE-Cy7), MHC-II (clone M5/114.15.2, Alexa Fluor 700), CD44 (clone IM7, PerCP-Cy5.5), CD127 (clone A7R34, FITC), CD25 (clone PC61.5, PerCP-Cy5.5), IL5 (clone TRFK5, PE), CD86 (clone GL1, PE), CD11c (clone N418, FITC), ST2 (clone RMST2-2, PE-Cy7) and FoxP3 (clone FJK-16S, APC); and SINFEKL from MBL international. tetramer (catalog number TB-5001-1, PE).

Human ILCs were defined as viable CD45 ⁺ , lineage ⁻ (CD3, CD5, CD56, CD11b, CD11c, CD16, CD19, TCRα/β, FcεR1), CD25 ⁺ , CD127 ⁺ cells as previously reported ⁷ . Human cells were stained with the following antibodies: BD Biosciences, GATA3 (clone L50-823, BV711), TBET (clone O4-46, BV650), RORγ-T (clone Q21-559, PE); Biolegend. , CRTH2 (clone BM16, PE-Cy7), CD11b (clone ICRF44, APC), CD56 (clone NCAM16.2, BV650), CD25 (clone BC96, PerCP-Cy5.5), CD45 (clone HI30, Pacific Blue), TCRα/β (clone IP26, APC); ThermoFisher Scientific, CD16 (clone CB16, APC), CD11c (clone 3.9, APC), CD127 (clone RDR5, FITC), CD3 (clone OKT3, Alexa Fluor 700) , ST2 (clone hIL33Rcap, PE), CD5 (clone L17F12, APC), CD19 (clone HIB19, AF700), FcεR1 (clone AER-37, APC). Human-specific antibody against IL33 (clone 390412, PE) was purchased from R&D Systems. All samples for flow cytometry were from prospectively collected unselected PDAC patients.

To examine intracellular cytokine production, single cell suspensions of tumors were treated with phorbol 12-myristate (PMA, PMA) in the presence of Brefeldin A (10 μg/ml) (all from Sigma-Aldrich). 100 ng/ml) and ionomycin (1 ng/ml) at 37° C. for 6 hours ex vivo. Cells were then surface stained, fixed, permeabilized and stained for cytokine production using the Fixation and Permeabilization Buffer kit according to the manufacturer's recommendations (Invitrogen, ThermoFisher Scientific). Appropriate isotype controls were used as indicated. Analyzes were performed with FlowJo (versions 9 and 10, Tree Star).

Immunohistochemistry Tissues were fixed with paraformaldehyde (Fisher Scientific) for 24 hours and embedded in paraffin. Tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), followed by antigen retrieval with CC1 buffer (Ventana Medical Systems). Sections were blocked with Background Buster solution (Innovex) for 30 minutes followed by avidin-biotin blocking for 8 minutes (Ventana Medical Systems). Mouse IL33 (AF3626, R&D Systems), mouse smooth muscle actin (Abcam), human IL33 (AF3625, R&D Systems) antibodies were applied and sections were incubated for 4 hours followed by a 1:200 dilution of biotinylated rabbit anti-goat IgG ( Vector labs) or biotinylated goat anti-rabbit IgG (Vector labs) for 60 minutes. Detection was performed using the DAB detection kit (Ventana Medical Systems) according to the manufacturer's instructions. All sections containing cells showing cytoplasmic or nuclear positivity for IL33 were designated as having positive staining. Slides were counterstained with Masson's trichrome or hematoxylin and eosin and coverslipped with Permount (Fisher Scientific). All tissue sections were evaluated by an independent PDAC pathologist.

immunofluorescence
Mouse IL33/CD11b/CK19/Iba1 immunofluorescence: Multiplex immunofluorescence staining was performed using the Discovery XT processor (Ventana Medical Systems) as described ²⁹ .
IL33: Sections were first incubated with anti-mIL33 (R&D Systems, Catalog No. AF3626, 1 μg/ml) for 4 hours, then biotinylated horse anti-goat IgG (Vector Laboratories) at a 1:200 dilution for 60 minutes. Detection was with streptavidin-HRP D (part of the DABMap kit, Ventana Medical Systems) followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen) prepared at predetermined dilutions according to the manufacturer's instructions.

CD11b: Sections were then incubated with anti-CD11b (Abcam, clone EPR1544) for 5 hours followed by biotinylated goat anti-rabbit IgG (Vector Laboratories) at 1:200 dilution for 60 minutes. Detection was with streptavidin-HRPD (part of the DABMap kit, Ventana Medical Systems) followed by incubation with Tyramide Alexa 594 (Invitrogen) prepared at predetermined dilutions according to the manufacturer's instructions.

CK19: Slides were then incubated with anti-CK19 (Abcam, clone EP1580Y) for 5 hours followed by a 1:200 dilution of biotinylated goat anti-rabbit (Vector Laboratories) for 60 minutes. Detection was performed with streptavidin-HRPD (part of the DABMap kit, Ventana Medical Systems) followed by incubation with Tyramide Alexa Fluor 546 (Invitrogen) prepared at predetermined dilutions according to the manufacturer's instructions.

Iba1: Finally, the sections were incubated with anti-Iba1 (Wako, catalog number 019-19741) for 5 hours, followed by a 1:200 dilution of biotinylated goat anti-rabbit IgG (Vector Laboratories) for 60 minutes. Detection was performed using streptavidin-HRPD (part of the DABMap kit, Ventana Medical Systems) followed by incubation with Tyramide Alexa 647 (Invitrogen) prepared at predetermined dilutions according to the manufacturer's instructions. rice field. After staining, slides were counterstained with DAPI (Sigma Aldrich) for 10 minutes and coverslipped with Mowiol.

Human tissue sections were deparaffinized with Leica Bond buffer (specially made by Leica Biosystems), and antigen retrieval was performed with Leica Bond ER2 buffer (Leica Biosystems). First, the sections were incubated with an anti-PD-1 antibody (Cell Marque, clone NAT105) for 1 hour and detected using a Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 488 (Invitrogen). Sections were then incubated with an anti-CD3 antibody (DAKO, catalog number A0452) for 1 hour and detected with a Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide CF594 (Biotum). Sections were then incubated with anti-GATA3 antibody (Cell Marque, clone L50-823) for 1 hour and detected with Bond Polymer Refine Detection kit (Leica Biosystems) and CF 543 (Biotum). Finally, the sections were incubated with an anti-CD45 antibody (DAKO, clone 2B11 + PD7/26) for 1 hour and detected with a Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 647 (Invitrogen). All detections were prepared at given dilutions according to the manufacturer's instructions. After staining, slides were counterstained with DAPI (Sigma Aldrich) for 10 minutes and coverslipped with Mowiol.

Digital Imaging and Analysis Slides were digitized using a Panoramic Flash 250 (3Dhistech, Budapest Hungary) with a Zeiss 20x/0.8NA objective and custom filters for A488, A546, A594 and A647. Each core was exported to a multichannel TIFF file and analyzed using a custom macro written in FIJI/ImageJ. For quantification, each nucleus was segmented using the DAPI channel after appropriate processing and background subtraction. Each nucleated cell was then evaluated for the presence or absence of other markers after setting an appropriate threshold for each marker. The number of cells with specific marker combinations was counted. ILC2 is CD45 ⁺ CD3 ⁻ GATA3 ⁺ nucleated cells, PD-1-expressing ILC2 is CD45 ⁺ CD3 ⁻ GATA3 ⁺ PD-1 ⁺ nucleated cells, PD-1-expressing T cells is CD45 ⁺ CD3 ⁺ PD-1 ⁺ Defined as nucleated cells. For each patient, the frequency of each cell type as a percentage of total nucleated cells was calculated in triplicate cores, then the average frequency of the triplicate cores was determined to calculate the final cell frequency per patient.

RNA sequencing mice: Tissues from orthotopic PDAC mice (n=6) were harvested and dispersed into single cell suspensions as described above. Tumor-infiltrating leukocytes were positively selected by magnetic-activated cell sorting using mouse CD45 MicroBeads (Miltenyi Biotec). Purification of magnetically activated sorted cells was confirmed by flow cytometry and was greater than 95%. RNA was isolated from sorted cells using the RNeasy Plus Mini Kit (Qiagen). Poly(A) capture and paired-end RNA-seq were performed by the MSKCC Integrated Genomics Core Facility. Specifically, after RiboGreen quantification and quality control on an Agilent BioAnalyzer, 500 ng of total RNA was subjected to polyA selection and TruSeq library preparation (TruSeq Stranded mRNA LT Kit, Cat. No. RS-122-2102) according to Illumina instructions. ) and 8 cycles of PCR were performed. Samples were barcoded and run on a HiSeq 4000 in 100bp/100bp paired-end runs using the HiSeq 3000/4000 SBS Kit (Illumina). An average of 83 million paired reads were generated per sample. Ribosomal reads accounted for up to 0.03% of all reads generated, with an average percentage of mRNA bases of 76.6%. Expression datasets were loaded into Gene Set Enrichment Analysis (GSEA) 3.0. The antigen presentation and T cell-mediated immunity gene set database was selected from MSIGDB v6.1, with a false discovery rate of 0.25 or less to facilitate exploratory discovery. GSEA was run with 1000 permutations. Three gene set databases met this threshold: GO 0002474 Antigen Processing and Presentation of Peptide Antigen Via MHC Class I, GO 0002711 Positive Regulation of T Cell Mediated Immunity, GSE19825 Naive vs Day 3 Effector CD8 T Cell Up.

Single-cell RNA sequencing Library preparation, sequencing, and post-processing of raw data for single-cell immune profiling were performed at the Epigenomics Core at Weill Cornell Medicine.

Single cell RNA library preparation and sequencing vehicle, IL33 alone, IL33 ⁺ PD-1 treated pancreatic KPC tumors and single cell suspensions of fluorescence activated cell (FAC) sorted ILC2 cells from mesenteric DLN was prepared as above. scRNA-seq libraries were generated according to 10X Genomics specifications (Chromium Single Cell V(D)J User Guide PN-1000006, 10x Genomics, Pleasanton, Calif., USA). Gel Beads-in-Emulsion (GEM) was generated by loading four independent cell suspensions (85-90% viability) at a concentration of 90-200 cells/μl onto the 10x Genomics Chromium platform, yielding approximately 2000 cells per sample. targeted single cells. After GEM generation, samples were incubated in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad, Hercules) for 45 min at 53° C. and Cell Barcodes and Template Switch Oligos conjugated to Unique Molecular Identifiers (UMI). (TSO) was added to generate a poly A cDNA with a barcode at the 5' end. GEM was cleaved and single-stranded cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, Waltham, Mass.). The cDNA was amplified for 16 cycles (98°C 45 seconds, 98°C 20 seconds, 67°C 30 seconds, 72°C 1 hour). cDNA quality was assessed using an Agilent Bioanalyzer 2100 (Santa Clara, Calif.), yielding a product of approximately 1,200 bp. 50 ng of cDNA was enzymatically fragmented, end-repaired into A tails, double-sided size selection with SPRIselect beads (Beckman Coulter, Indianapolis, Ind.), and ligated to the adapters included in the kit. . 14 cycles of PCR amplification using the index attached to the kit (98 ° C. 45 seconds, 98 ° C. 20 seconds, 54 ° C. 30 seconds, 72 ° C. 20 seconds × 14 cycles, 72 ° C. 1 minute, 4 ° C. hold), each live Introduced rally specific sample index. The indexed library was subjected to a second round of double-sided size selection, after which the library was quantified using Qubit fluorometry (Thermo Fisher Scientific, Waltham, Mass.). Quality was assessed with an Agilent Bioanalyzer 2100, resulting in an average library size of 450bp. None of the treated samples had concentrations below the limit of detection, 18 cycles of cDNA amplification were performed, and 16 cycles of sample indexing. Libraries were diluted to 10 nM and clustered using NovaSeq600 in paired-end read flow cells, R1 (10x barcode and UMI) for 28 cycles, I7 Index (sample index) for 8 cycles, R2 (transcript) at 89 bases. Sequencing yielded approximately 100 million clusters per sample (but tumors in vehicle-treated mice were approximately 10 million clusters), of which vehicle-treated tumor clusters accounted for 100 million clusters per sample. got a cluster. Primary processing of sequence images was performed using Illumina's Real Time Analysis software (RTA). Sample data were generated using the 10x Genomics Cell Ranger Single Cell Software suite v3.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger). Quality control using multiplexing, alignment to the mouse genome reference mm10, filtering, UMI count, single cell 5′ end gene count, and manufacturer parameters was performed. Approximately 11,000 single cells that passed quality control yielded data with an average of approximately 41,000 reads per cell (sequence saturation of 48%).

The Seurat R package version 3.1 pipeline was used to identify clusters of scRNA-seq data processing joint datasets ³⁰ . First, individual datasets were read into R as count matrices, converted to Seurat objects, and selected for genes expressed in ≥3 cells and cells in which at least 200 genes were detected. Cells were then sorted based on excluding cells with >2,500 or <200 unique genes expressed and cells with >5% mitochondrial gene content using a standard pretreatment workflow. Filtered.

After filtering, samples were pooled and gene expression measurements of retained cells were log-transformed, normalized by total expression per cell, and scaled by 10,000 molecules per cell. The top 2,000 genes with the highest variation across single cells were then identified and subjected to principal component (PC) analysis. After examining the jackstraw and elbow plots, K-nearest neighbor (KNN) clustering with cluster resolution set to 0.4 was used to select the top 15 PCs and all sample-bound and tumor-bound merged data 6-8 clusters were identified in the set. The dataset was also visualized using the top 15 PCs by non-linear dimensionality reduction using UMAP. Gene expression differences for gene marker searches between clusters were performed using the Wilcoxon rank sum test used in the Seurat package. To compare gene expression between samples, pairwise comparisons using the Wilcoxon rank sum test were performed with holm P-value adjustment.

In vitro assay
KPC 4662-GFP cells were plated in 96-well flat bottom plates (Falcon) in complete medium: 10% fetal bovine serum (Life Technologies), 100 units/ml penicillin, 100 μg/ml streptomycin, 0, 10, 100 and 500 ng/ml. Cultured for 1 week in RPMI-1640 containing L-glutamine (Gibco, ThermoFisher) plus ml concentration of recombinant IL33. Media and cytokines were replenished every 48 hours. Viability was measured using a colorimetric tetrazolium salt assay (Cell Counting Kit, Dojindo Molecular Technologies) according to the manufacturer's instructions and read on a Synergy HT Multi-Detection Microplate Reader (Biotek). Cells were harvested and stained for Annexin V (ThermoFisher Scientific), Ki-67 (clone SolA15, ThermoFisher Scientific) and ST2 (clone RMST2, ThermoFisher Scientific). For all in vitro studies, 2-3 technical replicates were performed for each independent study.

In Vitro Dendritic Cell Migration Assay Mouse splenic DC were isolated and enriched using Mouse Pan DC Isolation Kit (Miltenyi Biotech) according to the manufacturer's protocol. Flow cytometry was used to assess DC purity (>70% CD11c ⁺ of viable cells). Cells were seeded overnight at 5× ^{10 5} cells/ml complete RPMI medium supplemented with 50 ng/ml recombinant mouse GM-CSF (Biolegend). Chemotaxis of splenic DCs was then analyzed by a transwell migration assay. 600 μl of RPMI with or without 100 ng/ml recombinant mouse Ccl5 (Biolegend) was added to the lower chamber of a 6.5 mm Transwell plate with a 5.0-μm pore polycarbonate membrane insert (Sigma Aldrich). 200 μl of RPMI was also added to the upper chamber and the plate was equilibrated at 37° C. with 5% CO ₂ for 15 minutes. 1×10 ⁵ splenic DCs in 100 μl RPMI were then added to the upper chamber and incubated for 2 hours at 37° C. in 5% CO ₂ . After incubation, the membrane insert was carefully removed and cells were harvested from the lower chamber. Migrated DCs were incubated with DAPI and CD11c antibody for 20 min at 4° C., Precision Count Beads™ (Biolegend) were added, and live, migrated, and CD11c ⁺ cells were analyzed using flow cytometry according to the manufacturer's protocol. Cell numbers were quantified.

Statistical processing Data are presented as medians. We found many statistically significant effects in the data without calculating a pre-analytical sample size, so we did not use statistical methods to determine the sample size. Comparisons between two groups were performed with an unpaired Mann-Whitney test using a Benjamini-Krieger-Yekutieli false discovery approach for multiple time point comparisons (2-tailed). Comparisons between multiple groups were performed using a one-way ANOVA test followed by a Kruskal Wallis post-comparison test. Comparisons between multiple groups across multiple time points were performed using a two-way ANOVA test. Correlations between two variables were calculated using linear regression. Survival curves were compared by a two-tailed log-rank test. Tumor incidence was compared with the chi-square test. All α levels were 0.05 and P<0.05 was considered significant. Statistical analysis was performed using Prism 7.0 (GraphPad Software).

The information presented in this example was published by the inventors in the journal "Nature" (Moral et al., "ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity," Nature, 2020, Mar. , Vol. 579(7797), pp 130-135.doi: 10.1038/s41586-020-2015-4, Epub 2020 Feb 19). The entire contents of this publication, including supplementary material, are incorporated herein by reference.

Example 3
Effective treatment of PDAC in mice using IL33-activated ILC2 cell therapy
A study was conducted to evaluate the efficacy of IL33-activating ILC2 cell therapy using a mouse model of PDAC. Mice, PDAC models, active agents (IL33, αPD-1) and protocols were as described in Examples 1 and 2 above, unless otherwise stated. IL33 was administered to "donor" mice to activate pancreatic tumor ILC2 cells. Pancreatic tumor ILC2 cells were then isolated from donor mice, purified, and administered to "recipient" ILC2-deficient mice with established PDAC tumors. Some recipient mice were also given an anti-PD-1 antibody. Recipient mice receiving donor ILC2 cells alone had no significant effect on PDAC tumors, whereas recipient mice receiving donor ILC2 cells and anti-PD-1 antibody showed significant PDAC tumor size. confirmed to be decreasing.

Example 4
human clinical trials
Clinical trials are conducted to demonstrate the safety and/or efficacy of PDAC treatments. Clinical trials are conducted to demonstrate the safety and/or efficacy of treatment of PDAC with IL33 and/or PD-1/PD-L1 inhibitors in adult human subjects. Identify and enroll PDAC patients. Assign patients to specific groups. Different groups will receive either: (a) IL33 alone, (b) approved PD-1 and/or PD-L1 inhibitor alone, or (c) IL33 and approved PD-1. and/or receive a PD-L1 inhibitor. Within each group, there may be multiple different subgroups, such as with different drugs (eg, different PD-1/PD-L1 inhibitors), different dosages, and different dosing schedules. IL33 is administered intravenously (IV). In some subgroups, IL33 may be its native form. In some subgroups, IL33 may be modified by containing one or more half-life improving sites. Different subgroups may receive different doses of IL33 (within the range of possible doses). Different subgroups can receive IL33 at different frequencies (eg, daily or every 2, 3, 4, 5, 6, or 7 days). Approved PD-1 and/or PD-L1 inhibitors are administered at approved doses, by approved dosing schedules, and by approved routes of administration (e.g., as approved for the treatment of certain cancers). based on criteria).

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Claims (40)

A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering an effective amount of IL33 to a subject with PDAC, thereby treating the PDAC in the subject. 1. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering to a subject having PDAC an effective amount of (a) IL33 and (b) a PD-1 and/or PD-L1 inhibitor. administering, thereby treating PDAC in said subject. A method of activating pancreatic tissue-specific anti-tumor T cell immunity in a subject in need thereof, comprising administering an effective amount of IL33 to a subject with PDAC, thereby producing pancreatic tissue-specific anti-tumor T cells in said subject. A method comprising activating cellular immunity. A method of activating pancreatic tissue-specific anti-tumor T-cell immunity in a subject in need thereof, comprising an effective amount of (a) IL33 and (b) PD-1 and/or PD-L1 in a subject with PDAC and an inhibitor, thereby activating pancreatic tissue-specific anti-tumor T-cell immunity in said subject. A method of activating pancreatic ILC2 cells, comprising contacting the pancreatic ILC2 cells with an effective amount of IL33, thereby activating the pancreatic ILC2 cells. A method of activating pancreatic ILC2 cells, comprising contacting the pancreatic ILC2 cells with an effective amount of (a) IL33 and (b) a PD-1 and/or PD-L1 inhibitor, thereby activating the pancreatic ILC2 cells let, how. 7. The method of claim 5 or 6, wherein the ILC2 cells are contacted in vitro/ex vivo with IL33, PD-1 inhibitor or PD-L1 inhibitor. 7. The method of claim 5 or 6, wherein the ILC2 cells are contacted in vivo with IL33, PD-1 inhibitor or PD-L1 inhibitor. A method of sensitizing PDAC tumor and/or pancreatic ILC2 cells to PD-1 inhibitors and/or PD-L1 inhibitors comprising contacting the PDAC tumor and/or pancreatic ILC2 cells with an effective amount of IL33, A method, thereby sensitizing PDAC tumors and/or pancreatic ILC2 cells to PD-1 inhibitors and/or PD-L1 inhibitors. 5. The method of any one of claims 1-4, wherein the subject is a human. 5. The method of any one of claims 1-4, wherein the subject is a non-human mammal. 5. The method of any one of claims 1-4, wherein the subject is a mouse. 5. The method of any one of claims 1-4, wherein the subject has PDAC that is partially or totally resistant to PD-1 and/or PD-L1 inhibitor treatment. 10. The method of any one of claims 1-9, wherein the IL33 is recombinant IL33. 11. The method of claim 10, wherein IL33 is human IL33. 11. The method of claim 10, wherein IL33 is recombinant human IL33. 13. The method of claim 12, wherein IL33 is mouse IL33. 13. The method of claim 12, wherein IL33 is recombinant murine IL33. 10. The method of any one of claims 2, 4, 6 and 9, wherein the PD-1 inhibitor is an antibody. 10. The method of any one of claims 2, 4, 6 and 9, wherein the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, semiplimab, AMP-224, AMP-514 and PDR001. 10. The method of any one of claims 2, 4, 6 and 9, wherein the PD-L1 inhibitor is an antibody. 10. The method of any one of claims 2, 4, 6 and 9, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559 and CK-301. A pharmaceutical composition comprising (a) IL33 and (b) a PD-1 and/or PD-L1 inhibitor. A pharmaceutical composition for use in treating PDAC, comprising (a) IL33 and (b) a PD-1 and/or PD-L1 inhibitor. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof comprising: (a) administering to a recipient subject having PDAC an effective amount of activated donor pancreatic ILC2 cells, wherein the donor pancreas wherein the ILC2 cells are obtained from a donor subject and activated ex vivo/in vitro by contact with IL33, and the donor and recipient subjects are allogeneic, thereby treating PDAC in the recipient subject; Method. 1. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising: (a) contacting donor pancreatic ILC2 cells obtained from a donor subject ex vivo/in vitro with IL33 to activate donor pancreatic ILC2 cells; and (b) administering the donor-activated pancreatic ILC2 cells to a recipient subject with pancreatic ductal adenocarcinoma (PDAC), wherein the donor and recipient subjects are allogeneic, whereby A method comprising treating PDAC. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof comprising: (a) obtaining donor pancreatic ILC2 cells from a donor subject; (b) contacting the donor pancreatic ILC2 cells with IL33 ex vivo/in vitro. (c) administering the activated donor pancreatic ILC2 cells to a recipient subject with pancreatic ductal adenocarcinoma (PDAC), wherein the donor subject and recipient subject are allogeneic; , thereby treating PDAC in a recipient subject. 28. Any of claims 25-27, further comprising expanding the donor pancreatic ILC2 cells and/or the activated donor pancreatic ILC2 cells ex vivo/in vitro prior to administering the activated donor pancreatic ILC2 cells to the recipient subject. or the method described in paragraph 1. 29. The method of any one of claims 25-28, wherein the donor subject and recipient subject are the same person, wherein the method is autologous cell therapy. 29. The method of any one of claims 25-28, wherein the donor subject and recipient subject have the same MHC/HLA type. (none) 31. The method of any one of claims 25-30, wherein the donor and recipient subjects are humans. 31. The method of any one of claims 25-30, wherein the donor and recipient subjects are non-human mammals. 31. The method of any one of claims 25-30, wherein the donor and recipient subjects are mice. 35. The method of any one of claims 25-34, wherein the recipient subject has PDAC that is partially or totally resistant to PD-1 and/or PD-L1 inhibitor treatment. 36. The method of any one of claims 25-35, wherein the IL33 is recombinant IL33. 36. The method of any one of claims 25-35, wherein the donor and recipient subjects are human and the IL33 is human IL33. 36. The method of any one of claims 25-35, wherein the donor and recipient subjects are human and the IL33 is recombinant human IL33. 36. The method of any one of claims 25-35, wherein the donor and recipient subjects are mice and the IL33 is mouse IL33. 36. The method of any one of claims 25-35, wherein the donor and recipient subjects are mice and the IL33 is recombinant mouse IL33.

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